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WO2025155982A1 - Engineered transposases and methods of use thereof - Google Patents

Engineered transposases and methods of use thereof

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Publication number
WO2025155982A1
WO2025155982A1 PCT/US2025/012432 US2025012432W WO2025155982A1 WO 2025155982 A1 WO2025155982 A1 WO 2025155982A1 US 2025012432 W US2025012432 W US 2025012432W WO 2025155982 A1 WO2025155982 A1 WO 2025155982A1
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WO
WIPO (PCT)
Prior art keywords
amino acid
seq
acid positions
substitution
numbered
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/012432
Other languages
French (fr)
Inventor
David C. BAYS
Maura COSTELLO
Xiaoyu HUANG
Jack T. Leonard
Gavin John RUSH
Joseph C. Mellor
Jacob Hansen ARTZ
Anders Matthew KNIGHT
Kalyanaraman KRISHNAMOORTHY
Mathew G. MILLER
Aksiniya Lyubenova PETKOVA
Jonathan VROOM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Seqwell Inc
Original Assignee
Seqwell Inc
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Filing date
Publication date
Application filed by Seqwell Inc filed Critical Seqwell Inc
Publication of WO2025155982A1 publication Critical patent/WO2025155982A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1082—Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 69, 73, 91 , 127, 140, 144, 145, 146, 146/164, 150, 150/164, 151 , 153, 155, 156, 157, 160, 163, 164, 164/172, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/209, 165, 167, 169, 171 , 172, 182, 183, 188, 191 , 192, 193, 195, 196, 198, 199, 200, 204, 205, 208, 210, and 324, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
  • the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 77/156, 77/156/270, 77/184/244, 77/184/333, 77/244/270, 77/270, 143, 143/169/238/257/344, 143/169/255, 143/169/255/257, 143/169/343, 143/255, 143/255/257, 143/255/344, 143/257, 156, 156/270, 169/238, 169/238/255/257/344, 169/257/344, 169/344, 184, 184/244/324, 238, 238/255/344, 244/270, 257/344, and 270 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3662.
  • the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 77, 77/156/184/244/324, 77/184/270, 143, 143/238, 143/238/255, 143/238/255/343, 143/238/255/392, 143/238/344, 143/255, 143/257/392, 143/344, 169, 169/238/255/344, 169/238/344, 169/255, 169/343, 169/392, 238/344, 255, 255/257, 255/257/392, 257, and 344 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3662.
  • the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 20, 22, 26, 42, 43, 44, 45, 50, 51 , 52, 53, 54, 55, 60, 62, 64, 65, 95, 97, 99, 103, 104, 111 , 112, 115, 115/143/169, 115/169/343, 115/343, 116, 116/343, 143, 143/169, 154/439, 169, 169/324, 169/324/343, 169/343, 186, 234, 236, 238, 238/246, 239, 239/244, 241 , 242, 243, 244, 250, 251 , 254, 255, 260, 283, 284, 295, 296, 298/326, 319, 322, 323, 324, 324/343, 326, 328, 329, 333, 334, 338, 340, 341 ,
  • the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 26, 26/236, 26/236/260/333, 26/236/295, 26/260, 26/260/295, 26/260/295/333, 26/295/319, 26/333, 64, 64/244, 64/251/260/347, 64/260/295/333, 64/260/347, 103/115/255, 103/115/343, 103/115/343/344, 103/143/255/284/343/344, 103/169/343/344, 103/255/284, 103/343/344, 115, 115/143/255, 115/143/255/343, 115/143/284, 115/143/343, 115/143/344, 115/169, 115/169/343, 115/255, 115/255/344, 115/284, 115/284/343/344, 115/284/343, 115/284/343,
  • the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 115, 115/117, 115/117/118, 115/117/118/152, 115/117/118/169, 115/117/118/246, 115/117/152, 115/117/152/169, 115/117/152/169/246, 115/117/152/246, 115/117/169, 115/117/246, 115/118, 115/118/152, 115/118/152/169, 115/118/152/169/246, 115/118/169, 115/118/246, 115/152, 115/152/169, 115/152/169/246, 115/152/246, 115/152/246/374, 115/169, 115/169/246, 115/246, 117, 117/118, 117/118/152, 117/118/152/169, 117/118/152/246, 117/118/152/2
  • the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4642. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4642, wherein the C-terminal histidine tag is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4642, wherein the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof is replaced or removed.
  • the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 73, 101 , 114, 115, 118, 143, 145, 150, 151 , 152, 153, 154, 156, 157, 160, 161 , 163, 166, 167, 168, 169, 171 , 172, 190, 191 , 192, 195, 213, 214, 218, 219, 223, 225, 233, 233/343, 234, 235, 236, 237, 238, 239, 239/250, 243, 244, 249, 251 , 254, 255, 257, 258, 259, 261 , 262, 262/283, 277/292, 283, 284, 286, 289, 292, 295, and 296 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4278.
  • the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 93, 113, 113/114, 113/115, 113/143, 115/118, 115/177, 118/143/163/168/195/255/315, 118/143/255, 118/163/168/214/255/315, 118/168/214/315, 118/255, 124, 126, 129, 134, 143/154/255, 143/156/168/216/255, 143/163, 143/163/255, 143/168, 143/168/169/255/315, 143/255, 148, 156/214/315, 159, 163, 163/168, 163/168/169, 163/168/169/255/315, 163/168/169/315, 163/168/195/214/255/315, 163/214, 163/255, 163/255/315, 168/315, 186, 194, 197, 206
  • the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4872. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4872, wherein the C-terminal histidine tag is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4872, wherein the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4954.
  • the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4954, wherein the C-terminal histidine tag is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4954, wherein the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof is replaced or removed.
  • the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 127, 128, 129, 140, 180, 182, 194, 277, 293, 294, 299, 300, 317, 319, 320, 324, 333, 356, 366, and 392 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4642.
  • the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 113, 117, 118, 151 , 191 , 196, and 205 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4984.
  • the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 114/115/143, 114/115/143/233, 114/115/233, 114/115/343, 114/143, 114/143/151/233/238/261 , 114/143/213/261/343, 114/143/233, 114/143/233/238, 114/151/261/343, 114/213/238, 114/343, 115, 115/143, 115/143/151/154/213/261 , 115/143/151/154/233/343, 115/143/151/233/261 , 115/143/151/233/343, 115/143/154/213/233/238/261 , 115/143/213, 115/143/233, 115/143/343, 115/151/154/233, 115/151/261 , 115/213/233/238/261 , 115/143/213,
  • the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5354, wherein the C-terminal histidine tag is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5354, wherein the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof is replaced or removed.
  • the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 117, 117/118/143/168/169/392, 117/118/143/392, 117/118/168/169, 117/118/168/169/392, 117/143/168/169/170/392, 117/143/168/169/392, 117/168/169, 117/168/169/392, 117/168/392, 117/169, 117/392, 118/143, 118/143/168/169, 118/143/169, 118/143/392, 118/168, 118/168/392, 118/169/392, 118/392, 143/168/169/392, 143/169, 143/392, 168/169, 168/169/392, 168/392, 169/392, and 392 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to
  • the present invention provides engineered transposases capable of binding to adapters or donor DNA to form a transposome, cleaving or fragmenting target DNA, and/or ligating the adapters or donor DNA to the target DNA.
  • the engineered polypeptide comprises an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater sequence identity to any polypeptide sequence set forth in SEQ ID NOs: 2-2368, 2387-5694, and 5706-5756.
  • the engineered transposase comprises a polypeptide sequence selected from the polypeptide sequences between SEQ ID NOs: 2-2368, 2388-5694, and 5706-5756.
  • a functional fragment of an engineered polynucleotide sequence does not contain the segment of nucleotides encoding the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) and/or the 6-hisitine tag.
  • the altered nucleic acids are fragmented from the target nucleic acid.
  • the invention provides, a method of preparing a nucleic acid library comprising:
  • the engineered transposase comprises a polypeptide sequence comprising at least 85% sequence identity to a reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, or a functional fragment thereof, wherein the engineered transposase comprises at least one substitution or substitution set in its polypeptide sequence relative to the reference sequences;
  • the invention provides, a method of sequencing a target nucleic acid comprising:
  • the engineered transposase comprises a polypeptide sequence comprising at least 85% sequence identity to a reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, or a functional fragment thereof, wherein the engineered transposase comprises at least one substitution or substitution set in its polypeptide sequence relative to the reference sequence;
  • the method further includes adding a polynucleotide to the 5’ and/or 3’ end of each nucleic acid fragment of the plurality of nucleic acid fragments.
  • the engineered transposase is any one of the engineered transposases of the foregoing embodiments.
  • the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 2396.
  • the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4642.
  • the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4872.
  • the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4954.
  • the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5186.
  • the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5354. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5500. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5520. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5702. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5738.
  • the polypeptide sequence of the engineered transposase is modified to replace or remove the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) and/or the 6-histidine tag. In some embodiments, the polypeptide sequence of the engineered transposase is modified to replace the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) with a different peptide spacer. In some embodiments, the polypeptide sequence of the engineered transposase is modified to replace the 6-histidine tag with a different tag. In some embodiments, the polypeptide sequence of the engineered transposase is modified to remove the C-terminal histidine tag and the peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof.
  • the engineered transposase is purified.
  • fragmenting the target nucleic acid comprises tagmentation or random sheering and adapter ligation. In some embodiments, fragmenting the target nucleic acid comprises tagmentation.
  • each altered nucleic acid of the mixture of altered nucleic acids comprises an identifiable sequence tag (1ST).
  • each nucleic acid fragment of the subset of nucleic acid fragments comprises an identifiable sequence tag (1ST).
  • the 1ST is between 6 and 30 nucleotides in length (e.g., 6-10, 10-15, 15-20, 20-25, 25-30, such as 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).
  • each altered nucleic acid of the mixture of altered nucleic acids is between 30 and 30000 nucleotides in length (e.g., 30-30000, 30-25000, 30-20000, 30-15000, 30-10000, 30-7500, 30-5000, 30-4000, 30-3000, 30-2500, 30-2000, 30-1500, 30-1000, 30-900, 30-800, 30-700, 30-600, 30- 500, 30-400, 30-300, 30-250, 30-200, 30-150, 30-100, 30-50, 100-500, 100-1000, 100-1500, 100-2500, 100-5000, 100-10000, 100-20000, 100-30000, 500-1000, 500-2500, 500-5000, 500-10000, 500-20000, 500-30000, 1000-2500, 1000-5000, 1000-10000, 1000-20000, 1000-30000, 5000-10000, 5000-20000, 5000-30000, 10000-20000, 20000-30000, 2500-5000, 7500-10000, or 15000-25000 nucleotides in length).
  • each nucleic acid fragment of the plurality of nucleic acid fragments is between 30 and 30000 nucleotides in length (e.g., 30-30000, 30-25000, 30-20000, 30-15000, 30-10000, 30-7500, 30-5000, 30-4000, 30-3000, 30-2500, 30-2000, 30-1500, 30-1000, 30-900, 30-800, 30-700, 30- 600, 30-500, 30-400, 30-300, 30-250, 30-200, 30-150, 30-100, 30-50, 100-500, 100-1000, 100-1500, 100- 2500, 100-5000, 100-10000, 100-20000, 100-30000, 500-1000, 500-2500, 500-5000, 500-10000, 500- 20000, 500-30000, 1000-2500, 1000-5000, 1000-10000, 1000-20000, 1000-30000, 5000-10000, 5000- 20000, 5000-30000, 10000-20000, 20000-30000, 2500-5000, 7500-10000, or 15000-25000 nucleotides in length).
  • 30-50 100-500
  • the transposition event occurs in reaction conditions additionally comprising a terminal deoxynucleotide transferase, dNTP, and/or buffer components suitable for the addition of deoxynucleotides to the 3’ terminus of each of the plurality of nucleic acid fragments.
  • the transposition event occurs in reaction conditions additionally comprising a DNA ligase and/or buffer components suitable for a ligation reaction.
  • each altered nucleic acid of the mixture of altered nucleic acids further comprises a sample tag and/or a unique molecular identifier (UMI).
  • each nucleic acid fragment of the subset of nucleic acid fragments further comprises a sample tag and/or a unique molecular identifier (UMI).
  • amplification of the subset of nucleic acid fragments is performed through polymerase chain reaction (PCR), multiple displacement amplification (MDA), ligase chain reaction (LCR), loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), or strand displacement amplification (SDA).
  • PCR polymerase chain reaction
  • MDA multiple displacement amplification
  • LCR ligase chain reaction
  • LAMP loop mediated isothermal amplification
  • RCA rolling circle amplification
  • SDA strand displacement amplification
  • the sequencing comprises next-generation sequencing (NGS).
  • NGS next-generation sequencing
  • the sequencing comprises sequencing by synthesis, sequencing by ligation, or nanopore sequencing.
  • the sequencing by synthesis comprises IlluminaTM dye sequencing, single-molecule real-time (SMRTTM) sequencing, or pyrosequencing.
  • the sequencing by ligation comprises polony-based sequencing or SOLiDTM sequencing.
  • the target nucleic acid comprises genomic DNA or cDNAs from a single cell. In some embodiments, the target nucleic acid comprises nucleic acids from a plurality of haplotypes.
  • the target nucleic acid is crosslinked via histones or chromatin from single or multiple cells. In some embodiments, the target nucleic acid has been condensed or optionally treated with one or more condensing agents.
  • Figure 1 depicts active transposase variants, bound to appropriate adapter sequences, that are capable of binding target sequences, cleaving the target strand, and ligating the adapter oligonucleotides to the target fragment.
  • Figure 2 provides a scheme depicting the capillary electrophoresis (CE) assay used in the Examples to assess the activity of the engineered transposase variants.
  • CE capillary electrophoresis
  • Figure 3 is a plot of fragment sizes of NGS libraries prepared from 50 ng input DNA sample.
  • Figures 4A through 4D are plots showing the insertion bias of the indicated transposases as determined by the calculated root mean square error (RMSE) of the first 25 bases of the insert.
  • Figures 5A and 5B are gels showing the results of the enzyme activity assay comparing engineered transposase (SequenceK; Figure 5A) with a commercial transposase (hyperactive Tn5) for their ability to convert circular plasmid to linear form. 2.5 pmol transposase was used in the comparison. Plasmids are present in the gel as the higher length bars, i.e. >3000 bp, while the linear form fragments are shown as the smear ⁇ 3000 bp.
  • Figure 6 is a plot of fragment sizes of NGS libraries prepared from 50 ng - 200 ng input DNA sample.
  • Figure 7 is a plot showing the estimated NGS library size generated from 100 ng of human NA12878 genomic DNA using different engineered transposases (SequenceE, Sequencer, SequenceG, SequenceH, SequenceK, and SequenceL) and commercial hyperactive Tn5 transposases (Hyp-Tn5).
  • Figure 8 is a plot showing GC coverage bias of NGS libraries generated from 100 ng of human NA12878 genomic DNA using different engineered transposases (SequenceE; SequenceK; SequenceF; SequenceG; SequenceH; and SequenceL) and commercial hyperactive Tn5 transposases.
  • Figure 9 is a plot showing fold-coverage of NGS libraries generated from 100 ng of human NA12878 genomic DNA using different engineered transposases (SequenceE; SequenceK; SequenceF; SequenceG; SequenceH; and SequenceL) and commercial hyperactive Tn5 transposases.
  • Figures 10A through 10D are plots showing the per base sequence content of the transposition sites of the different commercial transposases and engineered transposases.
  • Each nucleotide (A, T, G, or C) is represented by a single line trace, representing the percentage of reads having that nucleotide at the corresponding position relative to the read start site (transposition site) of the DNA molecules in the sequenced library.
  • Figure 11 are plots showing GC coverage bias of NGS libraries prepared by ExpressPlex using hyperactive Tn5 transposase and the SequenceH engineered transposase from Staphylococcus epidermidis, Bacillus subtilis, Escherichia coll, and Pseudomonas aeruginosa.
  • Figure 12 is a plot showing the coverage profiles of NGS libraries prepared by ExpressPlex of lambda amplicon 18 DNA samples using a commercial and engineered (SequenceE, SequenceH, and SequenceK) transposases. Data is shown as the raw coverage profile as well as the summary statistic in the form of the coefficient of variation (CV) of the raw coverage profile.
  • Figure 13 is a plot showing the coverage profiles of NGS libraries prepared by ExpressPlex of pUC19 DNA samples using commercial and engineered (SequenceE, SequenceH, and SequenceK) transposases. Data is shown as the raw coverage profile as well as the summary statistic in the form of the coefficient of variation (CV) of the raw coverage profile.
  • ATCC refers to the American Type Culture Collection whose biorepository collection includes genes and strains.
  • amino acids are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.
  • a polynucleotide may contain one or more chemical modifications, including modifications on the 5’ end, the 3’ end, or internally.
  • chemical modifications include, but are not limited to, addition of functional groups (e.g., biotins, amino modifiers, alkynes, thiol modifiers, or azides), fluorophores (e.g. quantum dots or organic dyes), spacers (e.g. C3 spacer, dSpacer, photo-cleavable spacers), modified bases, or modified backbones.
  • polynucleotide oligonucleotide
  • nucleic acid is intended to comprise any modified or synthetic structure that is now known or discovered in the future that would be recognized by one of skill in the art as being or having the function of a “polynucleotide,” “oligonucleotide,” or “nucleic acid.”
  • An example of a modified or synthetic structure having the function of a “polynucleotide,” “oligonucleotide,” or “nucleic acid” is PNA or peptide nucleic acid.
  • Coding sequence refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.
  • the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • two polynucleotides or polypeptides may each (1 ) comprise a sequence ⁇ i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences
  • sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity.
  • a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence.
  • “Corresponding to,” “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.
  • the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence.
  • a given amino acid sequence such as that of an engineered transposase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences.
  • amino acid difference or “residue difference” refers to a change in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence.
  • the positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based.
  • Fragments refers to a polypeptide that has an amino-terminal and/or carboxyterminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence. Fragments can be at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long or longer, and up to 70%, 80%, 90%, 95%, 98%, and 99% of the full-length transposase polypeptide, for example the polypeptide of SEQ ID NO: 2 or a transposase provided in the polypeptide sequences of SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756.
  • the object species is purified to essential homogeneity (/.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules ( ⁇ 500 Daltons), and elemental ion species are not considered macromolecular species.
  • the isolated engineered transposase polypeptide is a substantially pure polypeptide composition.
  • Sequence insertion bias refers to the degree to which a transposome cleaves target nucleotide sequences in a non-random manner based on the composition of the sequence of the polynucleotide. For example, a transposase with G/C target bias will have an increased rate of binding to G/C rich sequences, as compared to other sequences. As detailed in the Examples below, sequence insertion bias can be approximated or quantified using several methods. One method evaluates bias over the first 15 nucleotides of NGS reads putatively comprising the region of the target bound by the transposome construct.
  • next generation sequencing refers to modern high-throughput or massively-parallel sequencing techniques that allow sequencing of many polynucleotide sequences at the same time, as compared to traditional sequencing methods, including the Sanger method, that allowed sequencing of only one polynucleotide at a time.
  • Next generation sequencing methods have facilitated whole genome sequencing and enabled genomics and related fields by requiring less polynucleotide sample and having greater accuracy.
  • Adapter refers to a donor polynucleotide that contains the end sequences for transposase binding, as well as other optional application-specific elements. These may include binding sites for sequencing primers, library amplification primers, sample indexes, or other functional donor polynucleotide sequence.
  • the adapter is loaded onto or bound to the transposase, which dimerizes to form the “transposome”.
  • “Complement” or “complementary,” as used herein in reference to a sequence refers to the sequence of a first nucleic acid in relation to that of a second nucleic acid, wherein when the first and second nucleic acids are aligned antiparallel (5’ end of the first nucleic acid matched to the 3’ end of the second nucleic acid, and vice versa) to each other, the nucleotide bases at each position in their sequences will have complementary structures following a lock-and-key principle (i.e. , A will be paired with U or T and G will be paired with C).
  • Complementary sequences may include mismatches of up to one third of nucleotide bases. For example, two sequences that are nine bases in length may have mismatches of at most 3, at most 2, or at most 1 , or at most 0 nucleotide bases, and remain complementary to one another.
  • Transposition or a “transposition event,” as used herein, is the cleavage of a target dsDNA (or hybrid DNA/RNA) sequence and ligation of the pre-loaded adapter (or another sequence containing transposon end sequences) from the transposome onto the target dsDNA at the site of cleavage.
  • transposition typically refers to the relocation of a segment of DNA to another locus in the genome.
  • transposition may result in the fragmentation of a target DNA sample with the simultaneous addition of the adapter sequences to the fragmented DNA ends, i.e., tagmentation.
  • “Synaptic complex” or “transposase synaptic complex,” as used herein, refers to a protein-nucleic acid complex including one or more transposases and one or more oligonucleotides.
  • the one or more oligonucleotides of the synaptic complex are inserted into a nucleic acid sequence of a nucleic acid sample by transposase activity.
  • the synaptic complex may include two transposases and two oligonucleotides.
  • the insertion of the two oligonucleotides into the nucleic acid sequence of the nucleic acid sample results in fragmentation of the nucleic acid sequence at the site of insertion.
  • the transposases may be Tn5 transposases.
  • the oligonucleotides may be adapter sequences.
  • the synaptic complex is pre-assembled.
  • “Increased enzymatic activity” refers to an improved property of the transposase polypeptides, which can be represented by an increase in specific activity ⁇ e.g., product produced/time/weight protein) or an increase in percent conversion of the substrate to the product ⁇ e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of transposase) as compared to the reference transposase enzyme. Exemplary methods to determine enzyme activity are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of K m , V max or k ca t, changes of which can lead to increased enzymatic activity.
  • Improvements in enzyme activity can be from about 1 .2 times the enzymatic activity of the corresponding wild-type enzyme, to as much as 2 times, 5 times, 10 times, 20 times, 25 times, 50 times or more enzymatic activity than the naturally occurring or another engineered transposase from which the transposase polypeptides were derived.
  • Transposase activity can be measured by any one of standard assays, such as by monitoring changes in properties of substrates, cofactors, or products.
  • the amount of products generated can be measured by Liquid Chromatography-Mass Spectrometry (LC-MS), HPLC, capillary electrophoresis or other methods, as known in the art.
  • Comparisons of enzyme activities are made using a defined preparation of enzyme, a defined assay under a set condition, and one or more defined substrates, as further described in detail herein. Generally, when lysates are compared, the numbers of cells and the amount of protein assayed are determined as well as use of identical expression systems and identical host cells to minimize variations in amount of enzyme produced by the host cells and present in the lysates.
  • Conversion refers to the enzymatic conversion of the substrate(s) to the corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzymatic activity” or “activity” of a transposase polypeptide can be expressed as “percent conversion” of the substrate to the product.
  • “Thermostable” refers to a polypeptide that maintains similar activity (more than 60% to 80% for example) after exposure to elevated temperatures ⁇ e.g., 40-80 °C) for a period of time ⁇ e.g., 0.5-24 hrs) compared to the wild-type enzyme exposed to the same elevated temperature.
  • solvent stable refers to a polypeptide that maintains similar activity (more than e.g., 60% to 80%) after exposure to varying concentrations ⁇ e.g., 5-99%) of solvent (ethanol, isopropyl alcohol, dimethylsulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time ⁇ e.g., 0.5-24 hrs) compared to the wild-type enzyme exposed to the same concentration of the same solvent.
  • solvent ethanol, isopropyl alcohol, dimethylsulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.
  • Thermo- and solvent stable refers to a polypeptide that is both thermostable and
  • T m melting temperature
  • the T m values for polynucleotides can be calculated using known methods for predicting melting temperatures (See e.g., Baldino et al., Meth. Enzymol., 168:761 -777 [1989]; Bolton et al., Proc. Natl. Acad. Sci.
  • the polynucleotide encodes the polypeptide disclosed herein and hybridizes under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered transposase enzyme of the present invention.
  • Hybridization stringency relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency.
  • the term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide.
  • Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in 0.2xSSPE, 0.2% SDS, at 42 °C.
  • High stringency hybridization refers generally to conditions that are about 10 °C or less from the thermal melting temperature T m as determined under the solution condition for a defined polynucleotide sequence.
  • a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCI at 65 °C (/.e., if a hybrid is not stable in 0.018M NaCI at 65 °C, it will not be stable under high stringency conditions, as contemplated herein).
  • High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in 0.1 xSSPE, and 0.1 % SDS at 65 °C.
  • Another high stringency condition is hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0.1% (w:v) SDS at 65 °C and washing in 0.1 x SSC containing 0.1 % SDS at 65 °C.
  • Other high stringency hybridization conditions, as well as moderately stringent conditions are described in the references cited above.
  • Heterologous polynucleotide refers to any polynucleotide that is introduced into a host cell by laboratory techniques, and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.
  • Codon optimized refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest.
  • the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome.
  • the polynucleotides encoding the transposase enzymes may be codon optimized for optimal production from the host organism selected for expression.
  • codons refers interchangeably to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid.
  • the preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression.
  • codon frequency e.g., codon usage, relative synonymous codon usage
  • codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene
  • multivariate analysis for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene
  • Codon usage tables are available for many different organisms (See e.g., Wada et al., Nucl. Acids Res., 20:2111 -2118 [1992]; Nakamura et al., Nucl. Acids Res., 28:292 [2000]; Duret, et al., supra; Henaut and Danchin, in Escherichia co// and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington D.C., p. 2047-2066 [1996]).
  • the data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein.
  • “Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed (/.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and/or polypeptide of interest.
  • “Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest.
  • the promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • Suitable reaction conditions refer to those conditions in the biocatalytic reaction solution ⁇ e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffers, co-solvents, etc.) under which a transposase polypeptide of the present invention is capable of converting one or more substrate compounds to a product compound ⁇ e.g., ligation or annealing of an adapter or donor polynucleotide and a target polynucleotide to form an annealed or ligated polynucleotide consisting of the adapter and the optionally fragmented target polynucleotide).
  • exemplary “suitable reaction conditions” are provided in the present invention and illustrated by the Examples.
  • composition refers to a mixture or combination of one or more substances, wherein each substance or component of the composition retains its individual properties.
  • a biocatalytic composition refers to a combination of one or more substances useful for biocatalysis.
  • “Loading”, such as in “compound loading” or “enzyme loading” or “cofactor loading” refers to the concentration or amount of a component in a reaction mixture at the start of the reaction. “Adapter loading” refers to binding of the transposase to the adapter polynucleotide.
  • “Substrate” in the context of a biocatalyst mediated process refers to the compound or molecule acted on by the biocatalyst.
  • a transposase biocatalyst used in the synthesis processes disclosed herein acts on an adapter or donor polynucleotide and a target polynucleotide.
  • Product in the context of a biocatalyst mediated process refers to the compound or molecule resulting from the action of the biocatalyst.
  • an exemplary product for a transposase biocatalyst used in a process disclosed herein is an annealed or ligated polynucleotide consisting of the adapter and the optionally fragmented target polynucleotide, as depicted in Figures 1 and 2.
  • Alkyl refers to saturated hydrocarbon groups of from 1 to 18 carbon atoms inclusively, either straight chained or branched, more preferably from 1 to 8 carbon atoms inclusively, and most preferably 1 to 6 carbon atoms inclusively.
  • An alkyl with a specified number of carbon atoms is denoted in parenthesis (e.g., (Ci-Ce)alkyl refers to an alkyl of 1 to 6 carbon atoms).
  • Alkenyl refers to hydrocarbon groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one double bond but optionally containing more than one double bond.
  • Alkynyl refers to hydrocarbon groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one triple bond but optionally containing more than one triple bond, and additionally optionally containing one or more double bonded moieties.
  • Heteroalkyl, “heteroalkenyl,” and heteroalkynyl refer respectively, to alkyl, alkenyl and alkynyl as defined herein in which one or more of the carbon atoms are each independently replaced with the same or different heteroatoms or heteroatomic groups.
  • Typical amino groups include, but are limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino, furanyl-oxy-sulfamino, and the like.
  • Aminoalkyl refers to an alkyl group in which one or more of the hydrogen atoms are replaced with one or more amino groups, including substituted amino groups.
  • aminocarbonyl refers to -C(O)NH 2 .
  • Substituted aminocarbonyl refers to -C(O)NR h R h , where the amino group NR h R h is as defined herein.
  • Oxy refers to a divalent group -O-, which may have various substituents to form different oxy groups, including ethers and esters.
  • Alkoxy or “alkyloxy” are used interchangeably herein to refer to the group -OR Z , wherein R z is an alkyl group, including optionally substituted alkyl groups.
  • Carboxy refers to -COOH.
  • Carbonyl refers to -C(O)-, which may have a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones.
  • Aminocarbonylalkyl refers to an alkyl substituted with an aminocarbonyl group, as defined herein.
  • Halogen or “halo” refers to fluoro, chloro, bromo and iodo.
  • Haloalkyl refers to an alkyl group in which one or more of the hydrogen atoms are replaced with a halogen.
  • haloalkyl is meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls.
  • Hydroalkyl refers to an alkyl group in which in which one or more of the hydrogen atoms are replaced with one or more hydroxy groups.
  • Thiol or “sulfanyl” refers to -SH. Substituted thiol or sulfanyl refers to -S-R h , where R h is an alkyl, aryl or other suitable substituent.
  • Optionally substituted refers to all subsequent modifiers in a term or series of chemical groups.
  • the term “optionally substituted arylalkyl,” the “alkyl” portion and the “aryl” portion of the molecule may or may not be substituted
  • Transposases with improved activity in cleaving or fragmenting polynucleotides and ligating or tagging polynucleotides are necessary to overcome the limitations of existing NGS library preparation methods. Improved transposases with increased polynucleotide fragmentation or cleavage activity, increased thermostability, increased activity at elevated temperatures, increased soluble expression, decreased inhibition, increased adapter loading or adapter ligation (i.e. tagging), increased binding to a target polynucleotide, and/or decreased sequence insertion bias compared to another wild-type or engineered transposase find utility in library preparation for NGS, but are not limited to these uses.
  • the improved transposases of the present disclosure find use in any application involving cleaving or fragmenting double-stranded polynucleotides and/or ligating adapters or tagging polynucleotides, including applications where a donor polynucleotide is transferred into a target polynucleotide.
  • the present invention provides novel transposases that have improved activity in the fragmentation or cleavage of double stranded DNA and the ligation of adapters or tagging of the DNA fragments.
  • the transposases of the present disclosure have increased polynucleotide fragmentation or cleavage activity, increased thermostability, increased activity at elevated temperatures, increased soluble expression, decreased inhibition, increased adapter loading or adapter ligation (i.e. tagging), increased binding to a target polynucleotide, and/or decreased sequence insertion bias as compared to another wild-type transposase or engineered transposase.
  • the engineered polypeptides of the present disclosure are variants of SEQ ID NO: 2, a chimeric protein based an IS4 transposase from Parashewanella curva or SEQ ID NO: 5704, a modified hyperactive Tn5 transposase, and designed to recognize the19 bp Tn5 mosaic end (ME) transposon donor recognition sequence.
  • These engineered transposases are capable of improved preparation of NGS libraries from polynucleotide samples.
  • transposases facilitate the transposition of fragments of DNA (transposons) from a donor strand to a host strand at sites flanked by short inverted-repeat sequences or end sequences (ESs).
  • the transposition mechanism is initiated by the binding of two transposases to donor DNA ESs to form the dimeric synaptic complex, which catalyzes cleavage of the donor DNA.
  • the transposome (dimeric transposase/DNA complex) then binds to the target DNA to transfer the transposon DNA to the target DNA.
  • Transposase-mediated NGS library preparation is similar to naturally- occurring transposition, except that the donor DNA or polynucleotide consists of two, separate adapter sequences rather than one continuous loop of donor DNA or polynucleotide. These adapter sequences are bound to transposases in a loading step that replaces the initial donor DNA or polynucleotide binding and cleavage step. Following adapter loading, the dimerized transposome proceeds to bind the target DNA or polynucleotide sample. Cleavage of the target polynucleotide and ligation of the adapter sequences results in fragmentation of the target DNA or polynucleotide and ligation of sequencing adapters to the target fragments.
  • further processing steps are used for library preparation, including gap filling, normalization, pooling of tagged fragment samples, and/or ligation of additional adapters to create dual-tagged libraries.
  • a variety of parameters may be manipulated to create sequencing libraries with various characteristics. For example, varying the concentration of transposase and adapters can change the fragment length. Copy number and sample coverage may also be influenced or controlled using methods known to those of skill in the art.
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2 and one or more residue differences as compared to SEQ ID NO: 2, selected from 10, 13, 24, 35, 39, 44, 46, 51 , 54, 55, 62/361 , 72, 75, 82, 89, 92, 107, 128, 137, 164, 167, 177/359, 191 , 192, 193, 194, 201 , 217, 281 , 289, 291 , 291/340, 305, 309, 310, 317, 331 , 337, 340, 346, 352, 353, 357, 358, 359, 362, 371 , 396, 414/435, 415,
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4 and one or more residue differences as compared to SEQ ID NO: 4, selected from 4, 5, 6, 8, 10, 10/317, 10/317/358, 10/317/359, 10/346, 10/346/359, 10/358, 10/359, 12, 12/373, 13, 15, 16, 18, 19, 26, 30, 30/154, 31 , 34, 35, 37, 38, 68, 70, 73, 74, 93, 106, 118, 126, 134, 135, 143, 146, 154/453, 163, 182, 207/286, 208, 226, 237, 246, 247, 259, 262, 263, 264, 280, 282, 286,
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4 and one or more residue differences as compared to SEQ ID NO: 4, selected from 4H, 5R, 6A, 8D, 10A, 10A/317W, 10A/317W/358V, 10A/317W/359A, 10A/346D, 10A/346D/359A, 10A/358V, 10A/359A, 12S, 12S/373C, 13V, 15S, 16S, 18A, 19L, 26A, 30N, 30N/154V, 31 V, 34Q, 35L, 37K, 38Y, 68A, 70A, 73K, 74A, 93E, 106E, 118S, 126V, 134
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 156 and one or more residue differences as compared to SEQ ID NO: 156, selected from 18/73/74/259, 18/73/286/289/411 , 18/74/118/259/286/289/411 , 18/74/259, 18/74/259/286/289, 18/74/259/286/411 , 18/118/259, 18/259/286/289/411 , 18/259/289/411 , 18/259/289/411 , 18/259/289/411 , 18/289/411 , 73/74/259/286/289, 73/74/259/289, 73/259/286, 74/259, 73
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 156 and one or more residue differences as compared to SEQ ID NO: 156, selected from 18A/73K/74A/259S, 18A/73K/286I/289P/411 K, 18A/74A/118S/259S/286I/289P/411 K, 18A/74A/259S,
  • 18A/74A/259S/286I/289P 18A/74A/259S/286I/289P, 18A/74A/259S/286I/411 K, 18A/118S/259S, 18A/259S/286I/289P/411 K, 18A/259S/289P/411 K, 18A/289P/411 K, 73K/74A/259S/286I/289P, 73K/74A/259S/289P, 73K/259S/286I, 74A/259S, 74A/259S/289P, 74A/286I/289P/411 K, 259S, 259S/286I/289P, and 289P.
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 302 and one or more residue differences as compared to SEQ ID NO: 302, selected from 16S/19L/355L/427A, 16S/37K/146M/286I/287N/289P/31 OQ/313R/355L, 16S/146M/226K/287N/289P/355L, 19L/37K/146M/286I/287N/310Q/355L, 19L/135R/286I/287N/289P/355L, 19L/182A/355L, 37K/70A/226K/31 OQ/313R/355L, 37K/74S/310Q/355L/427A, 37K/135R/146M/182A/355
  • K16S/C146M/E226K/T287N/K289P/C355L T19L/R37K/C146M/T286I/T287N/E310Q/C355L, T19L/K135R/T286I/T287N/K289P/C355L, T19L/S182A/C355L, R37K/V70A/E226K/E310Q/H313R/C355L, R37K/A74S/E310Q/C355L/S427A, R37K/K135R/C146M/S182A/E226K/T286I/T287N/C355L/A368S, R37K/C146M/E226K/K289P/S427A, R37K/C146M/T287N/K289P/S427A, C38F, C38N, K41 R, S42G
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from 12, 13, 18, 19, 22, 26, 317262, 34, 60, 79, 80, 81 , 82, 92, 106, 107, 113, 115, 132, 134, 136,
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from 12M, 131, 18Q, 19L, 22S, 22T, 26R, 31 M/262A, 34G, 60L, 79Y, 80T, 81 T, 82Q, 92L, 92T, 106Q, 107G, 107H, 107R, 113K, 115R, 132G, 132R, 134E, 134G, 136L, 140C, 140L
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from E12M, H13I, A18Q, T19L, P22S, P22T, K26R, T31 M/V262A, D34G, F60L, T79Y, A80T, V81T, L82Q, I92L, I92T, S106Q, D107G, D107H, D107R, S113K, K115R, F132G, F132R, T134E, T134G, T136L, I140C, I140L, 1140V, D143G, D143R, D143V, E152S, G160Q, G160T
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from S2A, E12L, E12M, H13I, H13N, H13Y, E15R, K16S, A18Q, T19L, P22S, P22T, K26R, E30H, T31 M/V262A, D34G, K51 R, F60L, E69A, E69L, T79Y, L82Q, Q84R, P87T, E88L, S106Q, D107G, D107H, D107R, S113E, V114P, K115S, L128F, F132G, F132R, T134
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from 44G/45V/47M/408R/453R, 44G/45V/47M/453H, 44G/45V/47M/453R/454R, 44G/47M/393A/453R/457G, 44S/45V/103M/453R/457G, 44S/45V/453T/454R/457G, 44S/47M/103M/393A/408R/453R/454R, 44S/47M/103M/453R/454R/457G, 44S/47M/103M/453R/454R/457G, 44S/47S/3
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from T44G/I45V/C47M/G408R/D453R, T44G/I45V/C47M/D453H, T44G/I45V/C47M/D453R/T454R, T44G/C47M/E393A/D453R/S457G, T44S/I45V/Q103M/D453R/S457G, T44S/I45V/D453T/T454R/S457G, T44S/C47M/Q103M/E393A/
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from 38F/45I/53D, 38F/45I/53D/54S/476S, 38F/45I/53L/244I, 38F/45I/53L/470N/472R, 38F/45I/54V/244T, 38F/45I/54V/470N/474S/478G, 38F/45I/472R/476S/478G, 38F/45I/476S, 38F/53D/54H/244I/472R/478G, 38F/53D/54S, 38F/53D/54S/244I, 38F/F/53D
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from C38F/V45I/M53D, C38F/V45I/M53D/Q54S/G476S, C38F/V45I/M53L/V244I, C38F/V45I/M53L/A470N/G472R, C38F/V45I/Q54V/V244T, C38F/V45I/Q54V/A470N/L474S/S478G, C38F/V45I/G472R/G476S/S478G, C38F/V45I/G478G, C
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from 22/38/45/107/259/317/412, 22/38/45/259/317/412, 22/38/64/134/220, 22/45/220/259, 22/64/107/259/317/412, 22/64/134/220, 22/107, 22/134, 22/134/317, 22/134/317/412, 22/220, 22/220/412/415, 22/259/317, 22/259/317/412, 22/317, 38, 38/45/81/143/244, 38/45/143/270, 38/53/243, 38
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from 22T/38F/45I/107R/259A/317M/412H, 22T/38F/45I/259A/317M/412H, 22T/38F/64R/134L/220R, 22T/45I/220R/259A, 22T/64R/107R/259A/317M/412H, 22T/64R/134L/220R, 22T/107R, 22T/134G, 22T/134G/317M/412H, 22T/134L/317M, 22T/134L/317M/412H, 22T/220R,
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 143, 143/175/272/273/415, 152/168/216/237, 152/168/423, 160, 160/201/272, 160/201/273/415, 160/226, 160/244/272/273/415, 160/415, 168, 168/187/216/255/423, 168/216/237, 168/255/423, 168/290/423, 187, 187/423, 199/201/226, 199/201/415, 201 , 216, 216/423, 226/244/415, 226/415, 2
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 143R, 143R/175T/272W/273P/415S, 152S/168Q/216E/237S, 152S/168Q/423D, 160Q, 160Q/201 G/272W, 160Q/201 G/273P/415S, 160Q/226R, 160Q/244I/272W/273P/415S, 160Q/415S, 168Q, 168Q/187G/216E/255R/423D, 168Q/216E/237S, 168Q/255R/423D, 168Q/290R/423D, 187G
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 143, 143/160/226/415, 143/175/272/273/415, 152/168/216/237, 152/168/423, 160, 160/201/272, 160/201/273/415, 160/226, 160/244/272/273/415, 160/415, 168, 168/187/216/255/423, 168/216/237, 168/216/237/255/343, 168/255/423, 168/290/423, 187, 199/201/226, 199/201/415, 201 , 216, 216/423
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 143R, 143R/160T/226R/415S, 143R/175T/272W/273P/415S, 152S/168Q/216E/237S, 152S/168Q/423D, 160Q, 160Q/201 G/272W, 160Q/201 G/273P/415S, 160Q/226R, 160Q/244I/272W/273P/415S, 160Q/415S, 168Q, 168Q/187G/216E/255R/423D, 168Q/216E/237S, 168Q/216E/237S/37S/
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from D143R, D143R/G160T/E226R/G415S, D143R/D175T/K272W/D273P/G415S, E152S/F168Q/D216E/D237S, E152S/F168Q/W423D, G160Q, G160Q/N201 G/K272W, G160Q/N201 G/D273P/G415S, G160Q/E226R, G160Q/V244I/K272W/D273P/G415S, G160Q
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 72, 90, 91 , 92, 93, 95, 122, 129, 141 , 142, 143, 144, 145, 150, 151 , 151/155, 152, 154, 155, 157, 160, 161 , 161/313, 164, 165, 168, 180, 181 , 184, 193, 197, 204, 205, 205/472, 206, 299, and 320.
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 72M, 72Q, 90F, 911, 91 V, 92A, 92L, 92V, 93A, 93H, 93S, 95G, 95S, 122F, 122L, 122Q, 122V, 129F, 1291, 141 Y, 142L, 143A, 143K, 144F, 1441, 144V, 144Y, 145F, 145L, 150D, 150E, 150G, 150R, 150S, 150T, 150V, 150W, 151 A, 151Q, 151 R, 151T
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 90, 92, 122, 129, 141 , 143, 144, 145, 150, 151 , 151/155, 152, 154, 155, 155/265, 157, 160, 161 , 164, 165, 168, 180, 181 , 184, 193, 197, 204, 205, 205/472, 206, 299, and 320.
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 90F, 92A, 92L, 92P, 92V, 122F, 122L, 129F, 1291, 141 Y, 143A, 143K, 144F, 144Y, 145F, 145L, 150D, 150E, 150G, 150R, 150S, 150V, 150W, 151 A, 151 K, 151Q, 151 R, 151T/155T, 152D, 152H, 152L, 152Q, 152R, 152T, 152V, 152Y, 154E, 154K,
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from L90F, I92A, I92L, I92P, I92V, W122F, W122L, L129F, L129I, H141 Y, D143A, D143K, W144F, W144Y, W145F, W145L, H150D, H150E, H150G, H150R, H150S, H150V, H150W, P151 A, P151 K, P151 Q, P151 R, P151 T/D155T, E152D, E152H, E152L, E152Q, E152R, E152T, E152V,
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1218 and one or more residue differences as compared to SEQ ID NO: 1218, selected from 129, 129/160, 129/160/164, 129/160/184, 129/164, 129/164/184, 129/205, 146/152/244, 150/155/226, 150/155/244, 152, 154, 154/160/184, 154/164, 154/205, 155, 160, 160/164, 160/164/184, 160/184, 164, 164/204, 164/205, 204, 205, 226, 226/244, and 244.
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1218 and one or more residue differences as compared to SEQ ID NO: 1218, selected from 1291, 1291/1601, 1291/1601/164Q, 1291/1601/184A, 1291/164Q, 1291/164Q/184A, 1291/2051, 146L/152T/244I, 150D/155T/226R, 150D/155T/244I, 152T, 154K, 154K/1601/184A, 154K/164Q, 154K/205I, 155T, 1601, 1601/164Q, 1601/164Q/184A, 1601/184A, 164Q, 164Q/204L, 164Q/205I,
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1218 and one or more residue differences as compared to SEQ ID NO: 1218, selected from L129I, L129I/G160I, L129I/G160I/D164Q, L129I/G160I/C184A, L129I/D164Q, L129I/D164Q/C184A, L129I/V205I, M146L/E152T/V244I, H150D/D155T/E226R, H150D/D155T/V244I, E152T, A154K, A154K/G160I/C184A, A154K/D164Q, A154K/V205I, D155T, G160
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1218 and one or more residue differences as compared to SEQ ID NO: 1218, selected from 1291, 1291/143H, 1291/154K/164Q, 1291/154K/164Q/205I, 1291/154K/205I, 1291/1601, 1291/1601/164Q, 1291/1601/2051, 1291/164Q/2051, 143R, 143R/150D/155T/226R/244I, 143R/150D/155T/244I, 143R/150D/226R, 143R/150D/226R/244I/343R, 143R/150D/226R/343R, 143R/150D/226R/3
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1218 and one or more residue differences as compared to SEQ ID NO: 1218, selected from L129I, L129I/D143H, L129I/A154K/D164Q, L129I/A154K/D164Q/V205I, L129I/A154K/V205I , L129I/G160I, L129I/G160I/D164Q, L129I/G160I/V205I, L129I/D164Q/V205I, D143R, D143R/H150D/D155T/E226R/V244I, D143R/H150D/D155T/V244I, D143R/H
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 98/129/157/168, 129/157/168, 151 , 151/152/255, 151/152/273, 151 /255, 151 /343, 157, 157/164, 157/164/168, 157/168, 164, 164/255, 168, 183, 255, 255/343, and 343.
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 981/1291/157R/168K, 1291/157R/168K, 151 Q, 151 Q/152T/255R, 151 Q/152T/273Y, 151 Q/255R, 151 R/343R, 157R, 157R/164D, 157R/164R, 157R/164R/168K, 157R/168K, 164D, 164D/255R, 168K, 183I, 255R, 255R/343R, and 343R.
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from L98I/L129I/K157R/Q168K, L129I/K157R/Q168K, P151 Q, P151 Q/E152T/A255R, P151 Q/E152T/D273Y, P151 Q/A255R, P151 R/E343R, K157R, K157R/Q164D, K157R/Q164R, K157R/Q164R/Q168K, K157R/Q168K, Q164D, Q164D/A255R, Q168K, V183I, A255R, A255R/E343
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 129, 129/157, 129/157/168, 129/164, 129/164/168/183, 129/164/183, 143, 143/151 , 143/151/152, 143/151/164, 143/164, 143/164/255, 143/164/343, 143/255, 143/343, 151 , 151/152, 151/152/164/343, 151/152/343, 151 /255, 151/255/343, 151/343, 152, 152/255, 157, 157/164/168, 157/
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 1291, 1291/157R, 1291/157R/168K, 1291/164R, 1291/164R/168K/183M, 1291/164R/183I, 143R, 143R/151 Q, 143R/151 R, 143R/151 R/152T, 143R/151 R/164D, 143R/164D, 143R/164D/255R, 143 R/164 D/343 R, 143R/255R, 143R/343R, 151 R, 151 R/152T, 151 R/152T/164D/343 R,
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from L129I, L129I/K157R, L129I/K157R/Q168K, L129I/Q164R, L129I/Q164R/Q168K/V183M, L129I/Q164R/V183I, D143R, D143R/P151 Q, D143R/P151 R, D143R/P151 R/E152T,
  • K157R/Q164R/Q168K K157R/Q168K, Q164D, Q164D/A255R, Q164R, Q164R/Q168K, Q164R/A447T, Q168K, V183I, A255R, A255R/E343R, V298I/E343R, and E343R.
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 69, 69/164, 72, 73, 91 , 92/164, 95, 96, 97, 98, 1 10, 1 12, 1 13, 121 , 121/164, 122, 122/164, 124, 126, 127, 139, 140, 141 , 144, 144/164, 145, 146, 147, 148, 150, 150/164, 151 , 151/164, 153, 153/164, 155, 156, 157, 159, 160, 161 , 163, 164, 164/172, 164
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 69Q, 69V/164D, 72L, 72Q, 73Q, 73W, 91 C, 91 L, 91 V, 92L/164D, 95G, 95S, 95V, 96G, 97T, 98V, 1 1 OR, 1 10T, 1 10V, 1 12A, 1 12H, 1 13V, 121 A, 121 L/164D, 121 Q, 122C, 122F, 122L, 122V, 122V/164D, 124L, 124M, 126A, 126S, 127
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from E69Q, E69V/Q164D, R72L, R72Q, K73Q, K73W, A91 C, A91 L, A91 V, I92L/Q164D, T95G, T95S, T95V, T96G, S97T, L98V, K1 10R, K1 10T, K1 10V, G1 12A, G1 12H, S1 13V, W121 A, W121 L/Q164D, W121 Q, W122C, W122F, W122L, W122V, W122V/Q164D, H124L
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from E69Q, E69V, E69V/Q164D, R72L, R72Q, K73Q, K73W, A91 C, A91 V, I92L/Q164D, T95S, S97T, L98M, L98V, W121 L/Q164D, T126A, T126S, L127I, L139I, I140L, 1140V, Q142C, W144F, W144L/Q164D, W144V, W144Y, W145F, W145K, W145L, M146A, M146F, M146K, M146L
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 73, 91 , 93, 95, 97, 98, 110, 112, 122, 122/164, 124, 127, 139, 144, 145, 146, 146/164, 147, 148, 150, 150/164, 151 , 151/164, 153, 153/164, 155, 156, 157, 159, 160, 161 , 162, 163, 164, 164/182, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/205/249, 164/20
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from K73Q, K73W, A91 C, A91 L, D93S, T95G, T95R, T95S, T95V, S97T, L98V, K1 10T, K1 10V, G1 12A, G1 12H, W122C, W122F, W122L, W122V, W122V/Q164D, H124A, H124L, H124M, L127I, L139I, W144F, W144Y, W145F, W145L, W145Q, W145R, W145S, W145T, M146A, M
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 69, 73, 91 , 127, 140, 144, 145, 146, 146/164, 150, 150/164, 151 , 153, 155, 156, 157, 160, 163, 164, 164/172, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/209, 165, 167, 169, 171 , 172, 182, 183, 188, 191 , 192, 193, 195, 196, 198, 199, 200,
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from 38/143/168/220/290/317/423, 38/152/154/164/168/220/290/317/423, 38/152/168/220/290/317/343/423, 38/152/168/220/290/317/423, 38/154/164/168/220/290/317/423, 38/168/220/290/317/423, and 38/220/317.
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from 38F/143R/168Q/220R/290R/317M/423D, 38F/152T/154K/164R/168K/220R/290R/317M/423D, 38F/152T/168Q/220R/290R/317M/343R/423D, 38F/152T/168Q/220R/290R/317M/423D, 38F/154K/164Q/168Q/220R/290R/317M/423D, 38F/168Q/220R/290R/317M/423D, and 38F/220R/317M.
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from C38F/D143R/F168Q/P220R/K290R/R317M/W423D,
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2368 and one or more residue differences as compared to SEQ ID NO: 2368, selected from 73Q/143K/1691/191 F/193W, 73Q/156R, 73Q/156R/210V, 73Q/156Y/192A/435K, 73Q/163D, 73Q/163D/210Q, 73Q/163N, 73Q/163N/210V, 73Q/192A/193W, 1271, 1271/160A/1721/198R, 1271/1721, 1271/198R, 146L, 146L/151 N/188G , 146L/324M, 151 N/171 V/324M,
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2426 and one or more residue differences as compared to SEQ ID NO: 2426, selected from 8, 13, 17, 18, 20/146/157/167, 26, 34/38, 38/39/45, 67, 70, 73, 73/150/155/169, 73/150/171/192, 73/155, 73/155/169/199, 73/155/171 , 73/192, 73/199, 78, 82, 84, 86, 86/384, 89, 90, 91 , 95, 102, 110, 111 , 113, 114, 115, 122/127, 126/127, 127/131/165
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2426 and one or more residue differences as compared to SEQ ID NO: 2426, selected from 8D, 8V, 13W, 17V, 18D, 18L, 18P, 20D/146C/157P/167L, 26R, 34H/38C, 38C/39F/45I, 38C/39P/45I, 67G, 70G, 73H, 73Q/150N/155L/169T, 73Q/150N/171 R/192A, 73Q/155L, 73Q/155L/169T/199L, 73Q/155L/171 R, 73Q/192A, 73Q/199L, 73R, 78
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2426 and one or more residue differences as compared to SEQ ID NO: 2426, selected from Q8D, Q8V, H13W, A17V, A18D, A18L, A18P, G20D/M146C/K157P/H167L, K26R, D34H/F38C, F38C/S39F/V45I, F38C/S39P/V45I, S67G, V70G, K73H, K73Q/H150N/D155L/C169T, K73Q/H150N/Q171 R/S192A, K73Q/D155L, K73Q/D155L/C169T/S199
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2426 and one or more residue differences as compared to SEQ ID NO: 2426, selected from 8D, 18L, 25G, 29F, 30R, 67G, 73H, 73Q/150N/155L/169T, 73Q/150N/155L/171 R, 73Q/150N/171 R/192A, 73Q/171 R, 73Q/192A, 73Q/199L, 74R, 82F, 82Q, 86A, 86F/384T, 86I, 113E, 115A, 115Q, 118S, 127L/128I, 143H, 150N, 150N/155L/169
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2426 and one or more residue differences as compared to SEQ ID NO: 2426, selected from Q8D, A18L, T25G, I29F, E30R, S67G, K73H, K73Q/H150N/D155L/C169T, K73Q/H150N/D155L/Q171 R, K73Q/H150N/Q171 R/S192A, K73Q/Q171 R, K73Q/S192A, K73Q/S199L, A74R, L82F, L82Q, Y86A, Y86F/A384T, Y86I, S113E, K115A, K115Q,
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from 66, 67, 70, 101 , 102, 103, 104, 106, 107, 108, 113, 114, 115, 117, 118, 143, 152, 154, 235, 236/251 , 237, 238, 242, 243, 246, 247, 249, 250, 251 , 256/352, 257, 259, 285, 286, 287, 288, 289, 290, 292, 293, 294, 295, 296, 322, 323, 326, 329, 330, 333, 339
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from 66T, 67V, 70A, 101 S, 101 T, 102R, 102S, 103V, 104T, 106A, 107P, 107S, 107V, 108M, 113G, 113R, 114A, 1141, 114P, 114S, 115L, 115R, 117A, 118K, 143E, 143G, 143R, 143V, 152R, 154E, 154F, 154Q, 235R, 236V/251 T, 237S, 238L, 242T,
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from V66T, S67V, V70A, K101 S, K101T, H102R, H102S, Q103V, V104T, S106A, D107P, D107S, D107V, L108M, S113G, S113R, V114A, V114I, V114P, V114S, K115L, K115R, K117A, A118K, D143E, D143G, D143R, D143V, T152R, K154E, K154F, K154Q, T235R, I236V/K251T, D237S,
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from 18, 18/113/270, 18/113/270/329/370, 18/113/270/370/384, 18/113/270/370/412, 18/113/329/384, 18/259/370/384, 18/270, 18/270/370, 18/270/370/384, 18/370, 18/370/384, 73, 73/115, 73/115/184/333, 73/115/184/341 , 73/115/256, 73/184/256, 73/184/273/333, 73/184/333, 73/256, 73/256,
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from 18D, 18D/113A/270L, 18D/113A/270L/329A/370S, 18D/259R/370S/384V, 18D/270L, 18D/270L/370S, 18D/270U370S/384V, 18D/370S, 18L, 18L/113A/270L, 18L/113A/270L/370S/384V, 18L/113A/329A/384V, 18L/113E/270L/370S/412S, 18L/370S, 18L/370S/384V, 73H, 73H
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from A18D, A18D/S113A/K270L,
  • A18D/S113A/K270L/K329A/T370S A18D/S259R/T370S/A384V, A18D/K270L, A18D/K270L/T370S, A18D/K270L/T370S/A384V, A18D/T370S, A18L, A18L/S113A/K270L, A18L/S113A/K270L/T370S/A384V, A18L/S113A/K329A/A384V, A18L/S113E/K270L/T370S/G412S, A18L/T370S, A18L/T370S/A384V, Q73H, Q73H/K115Q/C184A/R341 G, Q73H/C184A/R256G, Q73H/D273V/T333L/L375S, Q73H/T333L, Q73R, Q73R/K115Q, Q73R/
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from 18, 18/113, 18/113/252, 18/113/252/336/384, 18/113/252/370, 18/113/270, 18/113/270/370/384, 18/259/370/384, 18/270, 18/270/370, 18/270/370/384, 18/370, 18/370/384, 73, 73/115/184/341 , 73/131/256/273/333/341 /375, 73/184/256, 73/184/256/341 , 73/184/256/375, 73/184/256/256/256/341
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3662 and one or more residue differences as compared to SEQ ID NO: 3662, selected from 77, 77/156/184/244/324, 77/184/270, 143, 143/238, 143/238/255, 143/238/255/343, 143/238/255/392, 143/238/344, 143/255, 143/257/392, 143/344, 169, 169/238/255/344, 169/238/344, 169/255, 169/343, 169/392, 238/344, 255, 255/257, 255/257/392, 238/344, 255, 255/257,
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3662 and one or more residue differences as compared to SEQ ID NO: 3662, selected from 77E, 77E/156L/184A/244K/324M, 77E/184A/270P, 143E/238V, 143 E/238 V/255 R/343R, 143E/238V/255R/392P, 143E/238V/344M, 143E/257R/392P, 143E/344M, 143H, 143H/238V/255R, 143H/255R, 169T, 169T/238V/255R/344M, 169T/238V/344M, 169T/238V/344M, 169T
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3788 and one or more residue differences as compared to SEQ ID NO: 3788, selected from 20R, 22F, 22L, 22N, 22R, 22V, 22W, 26R, 42G, 42P, 43L, 43P, 43V, 44G, 44P, 44V, 45A, 45E, 45G, 45L, 45M, 45Q, 45R, 45W, 50R, 51 Q, 51 R, 52K, 52P, 52R, 52T, 53A, 53H, 53R, 53S, 54R, 54S, 54T, 54V, 55G, 55M, 55S, 55T, 55V, 60L, 62G, 64A, 64D, 64E
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4150 and one or more residue differences as compared to SEQ ID NO: 4150, selected from K26R, K26R/I236T, K26R/I236T/L260I/L333Y, K26R/I236T/L295I, K26R/L260I, K26R/L260I/L295I, K26R/L260I/L295I/L333Y, K26R/L295I/T319K, K26R/L333Y, P64A, P64A/V244T,
  • D143E/C169T/A255K/E284G/E343M/E344G D143E/C169T/E343M/E344G, D143E/A255K, D143E/A255K/E284A/E343M/E344G, D143E/A255K/E343M, D143E/A255K/E343M/E344G, D143E/A255K/E344G, D143E/E343M/E344G, C169T, C169T/A255K, C169T/A255K/E284G, C169T/A255K/E343M, C169T/E284A, C169T/E284G, C169T/E343M, C169T/E344G, I236T, I236T/V244T/L333Y, I236T/L260I, I236T/N347R, V244T, V244T
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4278 and one or more residue differences as compared to SEQ ID NO: 4278, selected from 115E, 115E/117A, 115E/117A/118T, 115E/117A/118T/152R, 115E/117A/118T/169T, 115E/117A/118T/246D, 115E/117A/152R, 115E/117A/152R/169T, 115E/117A/152R/169T/246D, 115E/117A/152R/246D, 115E/117A/152R/246D, 115E/117A/152R/246D, 115E/117A/169T, 115E
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4278 and one or more residue differences as compared to SEQ ID NO: 4278, selected from K115E, K115E/K117A, K115E/K117A/S118T, K115E/K117A/S118T/T152R, K115E/K117A/S118T/C169T, K115E/K117A/S118T/S246D, K115E/K117A/T152R, K115E/K117A/T152R/C169T,
  • K117A/S118T K117A/S118T, K117A/S118T/T152R, K117A/S118T/T152R/C169T, K117A/S118T/T152R/S246D, K117A/S118T/C169T, K117A/S118T/S246D, K117A/T152R, K117A/T152R/C169T,
  • K117A/T152R/S246D/S288G K117A/C169T, K117A/S246D, S118T/S246D, T152R, T152R/C169T, T152R/C169T/S246D, T152R/S246D, C169T, C169T/S246D, and S246D.
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4278 and one or more residue differences as compared to SEQ ID NO: 4278, selected from Q73L, Q73R, K101 S, V1 14S, K1 15A, K1 15Q, K1 15R, K1 15S, S1 18R, D143E, D143L, D143Q, D143V, W145Y, H150I, H150L, H150M, H150T, P151 A, P151 D, P151 F, P151 H, P151 R, T152L, D153G, K154P, K154S, K154Y, E156D, E156L, E156Q, E156W, K157L, K157P, K157R, A
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4642 and one or more residue differences as compared to SEQ ID NO: 4642, selected from 93, 1 13, 1 13/1 14, 1 13/1 15, 1 13/143, 1 15/1 18, 1 15/177, 1 18/143/163/168/195/255/315, 1 18/143/255, 1 18/163/168/214/255/315, 1 18/168/214/315, 1 18/255, 124, 126, 129, 134, 143/154/255, 143/156/168/216/255, 143/163, 143/163/255, 143/168, 143/168/169/255/315, 143/255, 148, 156/214/315
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4642 and one or more residue differences as compared to SEQ ID NO: 4642, selected from 93A, 93F, 93G, 93H, 93M, 93S, 93T, 1 13C/1 15K, 1 13G/1 15K, 1 13S, 1 13S/1 14G, 1 13S/1 15E, 1 13S/143K, 113S/143R, 1 15K/1 18C, 1 15K/1 18G, 1 15R/177V, 1 18T/143Q/163V/168N/195E/255K/315L, 1 18T/143Q/255K, 1 18T/163V/168N/214L/255K/315L
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4642 and one or more residue differences as compared to SEQ ID NO: 4642, selected from 127, 128, 129, 140, 180, 182, 194, 277, 293, 294, 299, 300, 317, 319, 320, 324, 333, 356, 366, and 392.
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5196 and one or more residue differences as compared to SEQ ID NO: 5196, selected from 117, 117/118/143/168/169/392, 117/118/143/392, 117/118/168/169, 117/118/168/169/392, 117/143/168/169/170/392, 117/143/168/169/392, 117/168/169, 117/168/169/392, 117/168/392, 117/168/392, 117/169, 117/392, 118/143, 118/143/168/169, 118/143/169, 118/143/169, 118/143/392,
  • K117R/S118R/M168K/V169T/C392P K117R/D143Q/M168K/V169T/R170H/C392A, K1 17R/D143Q/M168K/V169T/C392A, K1 17R/M168K/V169T, K117R/M168K/V169T/C392P,
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5286 and one or more residue differences as compared to SEQ ID NO: 5286, selected from T134P, T134R/S182T/P277W/E293G, T134R/S182T/P294Q, T134R/L194M/P277W, T134R/L194M/P277W/M317S, T134R/L194M/E293P, T134R/L194M/E293P/P294Q, T134R/L194M/E293P/M317S, T134R/L194M/P294Q, T134R/L194M/E293P
  • T134V/S182T/L194M/P294Q T134V/S182T/E293P/A320Y, T134V/L194M/P277W, T134V/L194M/E293P, S182T, S182T/L194M, S182T/L194M/E293G/A320Y, S182T/E293G/P294Q, L194M, L194M/P277W/E293G/M317S, L194M/P277W/M317S/A320Y, L194M/P277W/A320Y, L194M/E293P/P294Q/M317S, L194M/E293P/A320Y, P277W, E293P, M317S, M317S/A320Y, and A320Y.
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5412 and one or more residue differences as compared to SEQ ID NO: 5412, selected from 12/134, 13/134, 13/134/210, 22/134/152, 22/134/235/344, 64/93/134/235, 64/134/344/347, 77/134, 79/134/210, 84/134, 86/134, 87/134, 89/134, 93/134/152/235/344, 93/134/235/344, 93/134/244/344, 93/134/344, 131 , 134/152/344, 134/175, 134/210/382, 134/210/402
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5412 and one or more residue differences as compared to SEQ ID NO: 5412, selected from 12N/134R, 13C/134T/210R, 13L/134R, 13L/134T, 13R/134R, 13S/134R, 13T/134R, 13V/134R, 13V/134T/210R, 22R/134R/152P, 22R/134R/235S/344T, 64R/93T/134R/235S, 64R/134R/344Q/347R, 77Y/134R, 79V/134T/21 OR, 84V/134R, 86S/134R, 87G/134R, 87M/
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5412 and one or more residue differences as compared to SEQ ID NO: 5412, selected from E12N/P134R, H13C/P134T/H210R, H13L/P134R, H13L/P134T, H13R/P134R, H13S/P134R, H13T/P134R, H13V/P134R, H13V/P134T/H210R, P22R/P134R/T152P, P22R/P134R/T235S/E344T, P64R/D93T/P134R/T235S, P64R/P134R/E344Q/N347R, E77Y/
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5412 and one or more residue differences as compared to SEQ ID NO: 5412, selected from 73K/127L/134R/160G/172R/198Q/290R, 73K/127L/134R/160G/198Q/333T, 73K/127L/134R/172R/192S/266A/333T, 73K/127L/134R/172R/198Q, 73K/127L/134R/333T, 73K/127L/134T/160G/210R/266A, 73K/127L/134T/210R/290R, 73K/127L/134T/210R/333T, 73K/27L
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5412 and one or more residue differences as compared to SEQ ID NO: 5412, selected from Q73K/I127L/P134R/A160G/I172R/R198Q/K290R, Q73K/I127L/P134R/A160G/R198Q/L333T, Q73K/I127L/P134R/I172R/A192S/V266A/L333T, Q73K/I127L/P134R/I172R/R198Q, Q73K/I127L/P134R/L333T, Q73K/I127L/P134T/A160G/H210R/V266A,
  • the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5422 and one or more residue differences as compared to SEQ ID NO: 5422, selected from 13, 13/178/381 , 13/178/381/473, 13/178/457, 13/381 , 13/381/457, 13/402/457, 13/457, 178, 178/381/457, 178/381/457/473, 178/402/457, 178/473, 381 , 381/393/457, 381/457/473, 381/473, and 393/457.
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from
  • the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5704 and one or more residue differences as compared to SEQ ID NO: 5704, selected from 48V, 116E, 118E, 120R, 1301, 137R, 153H, 157K, 163A, 1751, 181 R, 187A, 192S, 213R, 246G, 263I, 273P, 296I, 318M, 334L, 341 L, 394A, 412N, 424D, 454R, and 458M.
  • the engineered transposase polypeptides are also capable of converting substrates (e.g., an adapter or donor polynucleotide and a target polynucleotide) to products (e.g., an annealed or ligated polynucleotide consisting of the adapter and the fragmented target polynucleotide).
  • substrates e.g., an adapter or donor polynucleotide and a target polynucleotide
  • products e.g., an annealed or ligated polynucleotide consisting of the adapter and the fragmented target polynucleotide.
  • the engineered transposase polypeptide is capable of converting the substrate compounds to the product compound with at least 1 .2 fold, 1 .5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, or more activity relative to the activity of the reference polypeptide of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704.
  • the engineered transposase has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, that increases soluble expression or isolated protein yield of the engineered transposase in a bacterial host cell, particularly in E. coli, as compared to a wild-type or engineered reference transposase, and comprises an amino acid sequence selected from the polypeptide sequences in SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756.
  • the engineered transposase has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, that increases the activity of the engineered transposase at high temperatures (by way of example and not limitation, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, or 65 °C), as compared to a wild-type or engineered reference transposase, and comprises an amino acid sequence selected from the polypeptide sequences in SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756.
  • the engineered transposase has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, that reduces the inhibition, as compared to a wild-type or engineered reference transposase, and comprises an amino acid sequence selected from the polypeptide sequences in SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756.
  • the engineered transposase has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, that increases specific activity of the engineered transposase on one or more an adapter or donor polynucleotide and target polynucleotide substrates as compared to a wild-type or engineered reference transposase, and comprises an amino acid sequence selected from the polypeptide sequences in SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756.
  • the engineered transposase with one or more improved properties has an amino acid sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from the polypeptide sequences of SEQ ID NOs: 2-2368, 2388-5694, and 5706-5756.
  • the engineered transposase with one or more improved properties has an amino acid sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704.
  • the engineered transposase with one or more improved properties has an amino acid sequence comprising a sequence selected from the polypeptide sequences of SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756. In some embodiments, the engineered transposase with one or more improved properties has an amino acid sequence comprising a sequence selected from SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704.
  • exemplary engineered transposases useful in the methods described herein comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13) polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to, comprising the amino acid sequences of, or consisting the amino acid sequences of SEQ ID NOs: 78, 146, 342, 746, 862, 894, 1006, 1668, 2444, 3680, 4782, 5234, 5436, and 5686.
  • polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
  • exemplary engineered transposases useful in the methods described herein comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13) polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to, comprising the amino acid sequences of, or consisting the amino acid sequences of SEQ ID NOs: 14, 68, 80, 92, 100, 506, 1004, 2090, 3456, 4562, 4674, 4682, 4790, 5002, and 5604.
  • polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities
  • exemplary engineered transposases useful in the methods described herein comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13) polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to, comprising the amino acid sequences of, or consisting the amino acid sequences of SEQ ID NOs: 58, 780, 1008, 1010, 1218, 1788, 2680, 2712, 2686, 3648, 4814, 5034, 5270, 5578, and 5688.
  • polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • exemplary engineered transposases useful in the methods described herein comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13) polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to, comprising the amino acid sequences of, or consisting the amino acid sequences of SEQ ID NOs: 14, 974, 1162, 1782, 2422, 2682, 4680, 4886, 4924, 5132, 5346, 5422, 5588, 5624, and 5680.
  • polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • exemplary engineered transposases useful in the methods described herein comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13) polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to, comprising the amino acid sequences of, or consisting the amino acid sequences of SEQ ID NOs: 5706-5756.
  • exemplary engineered transposases useful in the methods described herein comprise one or more polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to the amino acid sequences of SEQ ID NOs: 5706-5756, wherein the polypeptide sequence has at least one mutation selected from the group consisting of I48V, S116E, Q118E, K120R, L130I, F137R, D153H, A157K, G163A, R175I, S181 R, C187A, D192S, H213R, V246G, L263I, K273P, L296I, 1318M, T334L, Q341 L, E394A, K412N, W424D, E454R, and S458M.
  • any of the engineered transposase polypeptides disclosed herein can further comprise other residue differences relative to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, at other residue positions (i.e., residue positions other than those included herein). Residue differences at these other residue positions can provide for additional variations in the amino acid sequence without adversely affecting the ability of the polypeptide to carry out the conversion of substrate to product.
  • the amino acid sequence has optionally around 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 1 10, 120, 130, 140, or 150 amino acid residue deletions, insertions and/or substitutions.
  • the substitutions can be conservative or non-conservative substitutions.
  • non-encoded amino acids of which the polypeptides described herein may be comprised include, but are not limited to: the D-stereoisomers of the genetically- encoded amino acids; 2,3-diaminopropionic acid (Dpr); a-aminoisobutyric acid (Aib); e-aminohexanoic acid (Aha); 5-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine (Ocf); 3- chlorophenylalanine (Mcf); 4-
  • Non-encoding amino acids that are conformationally constrained of which the polypeptides described herein may be composed include, but are not limited to, N-methyl amino acids (L-configuration); 1 -aminocyclopent-(2 or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylic acid; homoproline (hPro); and 1 -aminocyclopentane-3-carboxylic acid.
  • the engineered polypeptides can be in various forms, for example, such as an isolated preparation, as a substantially purified enzyme, whole cells transformed with gene(s) encoding the enzyme, and/or as cell extracts and/or lysates of such cells.
  • the enzymes can be lyophilized, spray-dried, precipitated or be in the form of a crude paste, as further discussed below.
  • the engineered polypeptides can be in the form of a biocatalytic composition.
  • the polypeptides described herein are provided in the form of kits.
  • the enzymes in the kits may be present individually or as a plurality of enzymes.
  • the kits can further include reagents for carrying out the enzymatic reactions, substrates for assessing the activity of enzymes, as well as reagents for detecting the products.
  • the kits can also include reagent dispensers and instructions for use of the kits.
  • kits of the present invention include arrays comprising a plurality of different transposase polypeptides at different addressable position, wherein the different polypeptides are different variants of a reference sequence each having at least one different improved enzyme property.
  • a plurality of polypeptides immobilized on solid supports are configured on an array at various locations, addressable for robotic delivery of reagents, or by detection methods and/or instruments.
  • the array can be used to test a variety of substrate compounds for conversion by the polypeptides.
  • Such arrays comprising a plurality of engineered polypeptides and methods of their use are known in the art (See e.g., W02009/008908A2).
  • the present invention provides polynucleotides encoding the engineered transposase polypeptides described herein.
  • the polynucleotides may be operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide.
  • Expression constructs containing a heterologous polynucleotide encoding the engineered transposase are introduced into appropriate host cells to express the corresponding transposase polypeptide.
  • the present invention specifically contemplates each and every possible variation of polynucleotides that could be made encoding the polypeptides described herein by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 10.3, 10.5, 11 .2, 11 .4, 11 .5, 12.2, 12.3, 12.5, 12.6, 13.2, 13.3, 13.4, 14.2, 14.
  • the polynucleotide comprises a codon optimized nucleotide sequence encoding the transposase polypeptide amino acid sequence, as represented by SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704.
  • the polynucleotide sequence encodes at least one engineered transposase comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704.
  • the polynucleotide sequence comprises SEQ ID NOs: 1 , 3, 155, 301 , 337, 605, 971 , 1081 , 1217, 1579, 1597, 1599, 1603, 1719, 2367, 2425, 2539, 3401 , 3661 , 3787, 4149, 4277, 4641 , 4983, 5195, 5285, 5411 , 5421 , and/or 5703.
  • the polynucleotide encodes an engineered transposase polypeptide with improved properties as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150
  • the polynucleotides are capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from the polynucleotide sequences in the range of SEQ ID NOs: 1 -2367, 2387-5693, and 5705-5755 or a complement thereof, and encode a transposase polypeptide with one or more of the improved properties described herein.
  • the polynucleotide capable of hybridizing under highly stringent conditions encodes a transposase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, that has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150
  • the polynucleotides encode the polypeptides described herein but have at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding the engineered transposase.
  • the reference polynucleotide sequence is selected from SEQ ID NOs: 1 -2367, 2387-5693, and 5705-5755.
  • the polynucleotides encode the polypeptides described herein but have at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding the engineered transposase.
  • the reference polynucleotide sequence is selected from SEQ ID NOs: 1 -2367, 2387-5693, and 5705-5755.
  • control sequence is a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide.
  • the resultant polypeptide is referred to as a “proenzyme,” “propolypeptide,” or “zymogen,” in some cases).
  • a propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • regulatory sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems.
  • suitable regulatory systems include, but are not limited to the ADH2 system or GAL1 system.
  • suitable regulatory sequences include, but are not limited to the TAKA alphaamylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.
  • the polynucleotide sequence(s) of the present invention are expressed by inserting the polynucleotide sequence or a nucleic acid construct comprising the polynucleotide sequence into an appropriate vector for expression.
  • the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
  • the recombinant expression vector may be any vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and can result in the expression of the variant transposase polynucleotide sequence.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vectors may be linear or closed circular plasmids.
  • Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.
  • amdS acetamidase
  • argB ornithine carbamoyltransferases
  • bar phosphinothricin acetyltransferase
  • hph hygromycin phosphotransferase
  • niaD nitrate reductase
  • pyrG
  • the present invention provides a host cell comprising a polynucleotide encoding at least one engineered transposase polypeptide of the present invention, the polynucleotide being operatively linked to one or more control sequences for expression of the engineered transposase enzyme(s) in the host cell.
  • Host cells for use in expressing the polypeptides encoded by the expression vectors of the present invention are well known in the art and include but are not limited to, bacterial cells, such as E.
  • Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells typhimurium cells
  • fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris [ATCC Accession No. 201178]); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells.
  • Exemplary host cells are Escherichia co// strains (e.g., W3110 (AfhuA) and BL21 ).
  • the engineered transposases with the properties disclosed herein can be obtained by subjecting the polynucleotide encoding the naturally occurring or engineered transposase polypeptide to mutagenesis and/or directed evolution methods known in the art, and as described herein.
  • An exemplary directed evolution technique is mutagenesis and/or DNA shuffling (See e.g., Stemmer, Proc. Natl. Acad. Sci. USA 91 :10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651 ; WO 01/75767 and U.S. Pat. 6,537,746).
  • StEP staggered extension process
  • in vitro recombination See e.g., Zhao et al., Nat. Biotechnol., 16:258-261 [1998]
  • mutagenic PCR See e.g., Caldwell et al., PCR Methods Appl., 3:S136-S140 [1994]
  • cassette mutagenesis See e.g., Black et al., Proc. Natl. Acad. Sci. USA 93:3525-3529 [1996]).
  • mutagenesis and directed evolution methods can be readily applied to polynucleotides to generate variant libraries that can be expressed, screened, and assayed.
  • Mutagenesis and directed evolution methods are well known in the art (See e.g., US Patent Nos.
  • the enzyme clones obtained following mutagenesis treatment are screened by subjecting the enzymes to a defined temperature (or other assay conditions, such as testing the enzyme’s activity over a broad range of substrates) and measuring the amount of enzyme activity remaining after heat treatments or other assay conditions.
  • Clones containing a polynucleotide encoding a transposase polypeptide are then sequenced to identify the nucleotide sequence changes (if any), and used to express the enzyme in a host cell.
  • Measuring enzyme activity from the expression libraries can be performed using any suitable method known in the art (e.g., standard biochemistry techniques, such as HPLC analysis).
  • the polynucleotides encoding the enzyme can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be individually synthesized, then joined ⁇ e.g., by enzymatic or chemical ligation methods, or polymerase mediated methods) to form any desired continuous sequence.
  • polynucleotides and oligonucleotides encoding portions of the transposase can be prepared by chemical synthesis as known in the art (e.g., the classical phosphoramidite method of Beaucage et al., Tet. Lett.
  • oligonucleotides are synthesized ⁇ e.g., in an automatic DNA synthesizer), purified, annealed, ligated and cloned in appropriate vectors.
  • essentially any nucleic acid can be obtained from any of a variety of commercial sources.
  • additional variations can be created by synthesizing oligonucleotides containing deletions, insertions, and/or substitutions, and combining the oligonucleotides in various permutations to create engineered transposases with improved properties.
  • the amino acid sequence has optionally 1 -2, 1 -3, 1 -4, 1 -5, 1 - 6, 1 -7, 1 -8, 1 -9, 1 -10, 1 -15, 1 -20, 1 -21 , 1 -22, 1 -23, 1 -24, 1 -25, 1 -30, 1 -35, 1 -40, 1 -45, 1 -50, 1 -75, 1 -100, or 1 -150 amino acid residue deletions, insertions and/or substitutions.
  • the amino acid sequence has optionally around 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 1 10, 120, 130, 140, or 150 amino acid residue deletions, insertions and/or substitutions.
  • the substitutions can be conservative or non-conservative substitutions.
  • affinity techniques may be used to isolate the improved transposase enzymes.
  • any antibody which specifically binds the transposase polypeptide may be used.
  • various host animals including but not limited to rabbits, mice, rats, etc., may be immunized by injection with a transposase polypeptide, or a fragment thereof.
  • the transposase polypeptide or fragment may be attached to a suitable carrier, such as BSA, by means of a side chain functional group or linkers attached to a side chain functional group.
  • the affinity purification can use a specific ligand bound by the transposase or dye affinity column (See e.g., EP0641862; Stellwagen, “Dye Affinity Chromatography,” In Current Protocols in Protein Science, Unit 9.2-9.2.16 [2001]).
  • 29.2, 30.2, 31 .2, 32.2, 33.2, 34.2, 35.1 , 36.1 , and 36.3) is modified to replace the segment of nucleotides that encode the C-terminal 6-histidine tag with a different tag (e.g., a different affinity tag).
  • a different tag e.g., a different affinity tag.
  • the polynucleotide that encodes an engineered transposase e.g., any one of the engineered transposases described herein, such as in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 10.3, 10.5,
  • 30.2, 31 .2, 32.2, 33.2, 34.2, 35.1 , 36.1 , and 36.3) is modified to encode an engineered transposase with one or more additional tags (e.g., one or more affinity tags), such as the C-terminal 6-histidine tag and one or more additional tags.
  • tags such as affinity tags (e.g., affinity tags suitable for protein purification) are known in the art and include, for instance, a polyhistidine tag (e.g., five or more histidine residues), a polyarginine tag (e.g., five or more arginine residues), and a FLAG tag.
  • 29.2, 30.2, 31 .2, 32.2, 33.2, 34.2, 35.1 , 36.1 , and 36.3) is modified to replace the segment of nucleotides that encodes the C-terminal GGSG (SEQ ID NO: 5777) peptide spacer with a different peptide spacer.
  • Suitable peptide spacers are known in the art and comprise flexible amino acid residues such as glycine, alanine, and serine.
  • a peptide spacer includes 2 to 12 amino acids including motifs of GA or GS, e.g., GA, GS, GAGA (SEQ ID NO: 5787), GSGS (SEQ ID NO: 5788), GAGAGA (SEQ ID NO: 5789), GSGSGS (SEQ ID NO: 5790), GAGAGAGA (SEQ ID NO: 5791 ), GSGSGSGS (SEQ ID NO: 5792), GAGAGAGAGA (SEQ ID NO: 5793), GSGSGSGSGS (SEQ ID NO: 5794), GAGAGAGAGAGA (SEQ ID NO: 5795), and GSGSGSGSGSGSGS (SEQ ID NO: 5796).
  • a peptide spacer is cleavable. In some embodiments, a peptide spacer contains a protease binding site and can be cleaved by a protease via site-specific enzyme activity.
  • an engineered transposase (e.g., an engineered transposase of the invention) is modified to include one or more tags and/or linkers on either the N- or C- termini.
  • a tag e.g., a polyhistidine tag such as a 6-histidine tag
  • a portion of a peptide spacer of an engineered transposase e.g., an engineered transposase of the invention
  • the tag e.g., an affinity tag
  • a portion of the peptide spacer is removed, e.g., via proteolytic cleavage.
  • a polynucleotide that encodes an engineered transposase e.g., any one of the engineered transposases described herein, such as in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 10.3, 10.5, 11.2, 11.4, 11.5, 12.2, 12.3, 12.5, 12.6, 13.2, 13.3, 13.4, 14.2, 14. 3, 14.5, 14.6, 14.8, 14.9, 15.1 ,
  • the engineered polypeptide may be added to the reaction mixture in the form of a purified enzyme, partially purified enzyme, whole cells transformed with gene(s) encoding the enzyme, as cell extracts and/or lysates of such cells, and/or as an enzyme immobilized on a solid support.
  • Whole cells transformed with gene(s) encoding the engineered transposase enzyme or cell extracts, lysates thereof, and isolated enzymes may be employed in a variety of different forms, including solid (e.g., lyophilized, spray-dried, and the like) or semisolid (e.g., a crude paste).
  • the gene(s) encoding the engineered transposase polypeptides can be transformed into host cell separately or together into the same host cell.
  • one set of host cells can be transformed with gene(s) encoding one engineered transposase polypeptide, and another set can be transformed with gene(s) encoding another transposase. Both sets of transformed cells can be utilized together in the reaction mixture in the form of whole cells, or in the form of lysates or extracts derived therefrom.
  • a host cell can be transformed with gene(s) encoding multiple engineered transposase polypeptides.
  • the engineered polypeptides can be expressed in the form of secreted polypeptides, and the culture medium containing the secreted polypeptides can be used for the transposase reaction.
  • the improved activity of the engineered transposase polypeptides disclosed herein provides for processes wherein higher percentage conversion can be achieved with lower concentrations of the engineered polypeptide.
  • the suitable reaction conditions comprise an engineered polypeptide amount of about 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 20% (w/w), 30% (w/w), 40% (w/w), 50% (w/w), 75% (w/w), 100% (w/w) or more of substrate compound loading.
  • the engineered polypeptide is present at a molar ratio of engineered polypeptide to substrate of about 50 to 1 , 25 to 1 , 10 to 1 , 5 to 1 , 1 to 1 , 1 to 5, 1 to 10, 1 to 25 or 1 to 50. In some embodiments, the engineered polypeptide is present at a molar ratio of engineered polypeptide to substrate from a range of about 50 to 1 to a range of about 1 to 50.
  • a PCR reaction generates the amplicons of the sequence library by using the amplifier oligonucleotides as primers.
  • the amplicons are generated in a PCR reaction with a first universal primer and a second universal primer, wherein the first universal primer and the second universal primer bind to respective complementary universal primer regions in the sequencing oligonucleotides.
  • the amplicons may be further amplified in a PCR reaction with a first universal primer and a second universal primer, wherein the first universal primer and the second universal primer bind to respective complementary universal primer regions in the amplicons.
  • the amplicons include (a) a nucleic acid sequence including a first universal primer region, a first amplifier barcode region (e.g., a pool tag, e.g., an i5 or an i7 tag), a first amplifier priming region, a homologous sequence of a first nucleic acid fragment, a complement sequence of the second amplifier priming region, a complement sequence of the second amplifier barcode region (e.g., a pool tag, e.g., an i5 or an i7 tag), and a complement sequence of the second universal primer region; and (b) the complement sequence thereof.
  • a nucleic acid sequence including a first universal primer region, a first amplifier barcode region e.g., a pool tag, e.g., an i5 or an i7 tag
  • a first amplifier barcode region e.g., a pool tag, e.g., an i5 or an i7 tag
  • a first amplifier priming region e.g
  • a nucleic acid duplex including the first nucleic acid fragment and its complement is generated from the nucleic acid sample by transposase activity.
  • the intermediate nucleic acid is the template for the PCR reaction.
  • the PCR reaction produces amplicons from the intermediate nucleic acids using the amplifier oligonucleotides.
  • the PCR reaction includes 1 -35 cycles (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 cycles). In some instances, the PCR reaction includes more than 35 cycles.
  • the PCR reaction may be monitored by tracking the cycle threshold (Ct) via fluorescence detection using well- established methods in the art.
  • the resulting sequence library following transposition, tagmentation, and amplification may be evaluated for quality (e.g., size distribution, purity, concentration, among other quality considerations).
  • the sequence library may be assessed for the size distribution of the resulting fragments by gel electrophoresis compared to a reference sample (e.g., a DNA ladder).
  • one or more enzymes are inactivated by the application of heat or an inhibitor following a chemical reaction (e.g., transposition, polymerization, or amplification) in the tagmentation-based sequence library preparation.
  • a chemical reaction e.g., transposition, polymerization, or amplification
  • one or more enzymes are inactivated by changes in buffer or solvent condition (e.g., changes in pH or changes in salt concentration).
  • the nucleic acid sample is purified or partially purified following a chemical reaction.
  • the nucleic acid sample is concentrated or diluted following a chemical reaction.
  • one or more enzymes are immobilized to improve reaction efficiency, enzyme activity, or sample recovery.
  • an enzyme is immobilized on a solid support, a surface, a resin, a membrane, a bead, a nanoparticle, a biological cofactor (e.g., a protein, an antibody or antibody fragment, a peptide, an oligonucleotide, an aptamer), or a combination thereof.
  • a biological cofactor e.g., a protein, an antibody or antibody fragment, a peptide, an oligonucleotide, an aptamer
  • an enzyme is immobilized through an irreversible or a reversible reaction.
  • an enzyme is immobilized by cross-linking, adsorption, entrapment, encapsulation, click chemistry reactions, ionic bonding, covalent bonding, hydrophobic interactions, among other non-limiting methods of immobilization.
  • the nucleic acid sample, the first adapter oligonucleotide, the second adapter oligonucleotide, the first amplifier oligonucleotide, the second amplifier oligonucleotide, the intermediate nucleic acids, and/or the amplicons may include DNA.
  • the nucleic acid sample may include double-stranded DNA.
  • further processing steps are used for library preparation, including gap filling, normalization, pooling of tagged fragment samples, and/or ligation of additional adapters to create dualtagged libraries.
  • a variety of parameters may be manipulated to create sequencing libraries with various characteristics. For example, varying the concentration of transposase and adapters can change the fragment length. Copy number and sample coverage may also be influenced or controlled using methods known to those of skill in the art.
  • nucleic acid sequencing e.g., next-generation sequencing (NGS)
  • NGS next-generation sequencing
  • sequencing can be performed by various systems that are currently available, e.g., a sequencing system by Pacific Biosciences (PACBIO®), ILLUMINA®, Oxford NANOPORE®, Genapsys®, or ThermoFisher (ION TORRENT®).
  • PACBIO® Pacific Biosciences
  • ILLUMINA® Oxford NANOPORE®
  • Genapsys® Genapsys®
  • ION TORRENT® ThermoFisher
  • sequencing may be performed using nucleic acid amplification, sequencing by ligation (e.g., SOLiD or polony-based sequencing), sequencing by synthesis (e.g., Illumina dye sequencing, single-molecule real-time (SMRT) sequencing, or pyrosequencing), nanopore sequencing, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR), or isothermal amplification.
  • ligation e.g., SOLiD or polony-based sequencing
  • sequencing by synthesis e.g., Illumina dye sequencing, single-molecule real-time (SMRT) sequencing, or pyrosequencing
  • nanopore sequencing e.g., polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR), or isothermal amplification.
  • PCR polymerase chain reaction
  • the sequencing oligonucleotides and amplicons described herein can be uniquely identified based on the nucleic acid sequences of the nucleic acid fragment and the nucleic acid sequences of the adapter barcode regions (e.g., an identifiable sequence tag (1ST) or a sample tag) or amplifier barcode regions (e.g., pool tags, e.g., i5 or 17 tags) of the sequencing oligonucleotides or amplicons, respectively.
  • the invention further includes data generated by nucleic acid sequencing, as well as methods for generating and analyzing such sequence data, and reaction mixtures used in and formed by such methods.
  • Methods described herein can be used for assaying transposition activity of a transposase described herein (e.g., an engineered transposase described herein.
  • the methods include performing a transposition reaction on a nucleic acid (e.g., DNA, e.g., double-stranded DNA (dsDNA)), i.e. , to insert a nucleic acid sequence of interest (e.g., DNA, e.g., dsDNA, e.g., dsDNA adapters) into a target nucleic acid sequence (e.g., DNA, e.g., dsDNA).
  • a nucleic acid e.g., DNA, e.g., double-stranded DNA (dsDNA)
  • a nucleic acid sequence of interest e.g., DNA, e.g., dsDNA, e.g., dsDNA adapters
  • target nucleic acid sequence e.g.
  • 1 - 50 pM (e.g., 1 -40, 1 -30, 1 -20, 1 -10, 1 -5, 5-50, 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20, 20-50, 20-40, or 20-30 pM; e.g., about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 pM) of the adapter may be incubated with engineered transposases for about 60 minutes to load the transposases and generate synaptic complexes.
  • 1 - 50 pM e.g., 1 -40, 1 -30, 1 -20, 1 -10, 1 -5, 5-50, 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20, 20-50, 20-40, or 20-30 pM; e.g., about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14,
  • a target substrate e.g., a target dsDNA
  • a target substrate is added and incubated with the synaptic complexes until a transposition event occurs.
  • the mixture of the synaptic complexes and the target substrate is incubated (e.g., heated in a thermocycler).
  • the mixture is incubated at about 55 °C for 5-15 minutes (e.g., about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 minutes).
  • the target substrate is any of the nucleic acid samples described herein, including DNA or cDNA, and may be prokaryotic, eukaryotic, or artificial in origin.
  • the activity of two different transposases may be compared by comparing the amount of transposition reactions that occur when using synaptic complexes comprising the first transposase versus synaptic complexes comprising the second transposase.
  • the other reaction conditions are maintained similar or identical for the comparison (e.g., target nucleic acid concentration, reaction temperature, reaction duration, adapter concentration, transposase concentration, buffer conditions).
  • Methods described herein can be used for assaying transposition performance of a transposase employed in the methods (e.g., an engineered transposase described herein.
  • the methods include generating an NGS library from a nucleic acid sample (e.g., DNA, e.g., double-stranded DNA (dsDNA)), i.e., by performing a tagmentation reaction (e.g., to fragment the nucleic acid sample and insert adapter nucleic acids DNA, e.g., dsDNA, e.g., dsDNA adapters) and amplifying the nucleic acid fragments resulting from the tagmentation reaction.
  • a nucleic acid sample e.g., DNA, e.g., double-stranded DNA (dsDNA)
  • a tagmentation reaction e.g., to fragment the nucleic acid sample and insert adapter nucleic acids DNA, e.g., dsDNA, e.g.
  • synaptic complexes i.e., transposomes
  • transposases e.g., engineered transposases described herein
  • nucleic acid adapters e.g., DNA adapter oligonucleotides, e.g., i5 and i7 oligonucleotides
  • a nucleic acid sample is mixed with the synaptic complexes (e.g., loaded i5 and 17 transposases) and incubated until a transposition reaction occurs.
  • the mixture is incubated at about 55 °C for 5-15 minutes (e.g., about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 minutes).
  • the transposition reaction is performed using a method described herein.
  • the product of the transposition reaction is amplified, e.g., using a method of nucleic acid amplification known in the art (e.g., a PCR reaction, e.g., using primers suitable for the i5 and I7 adapter sequences).
  • the nucleic acid sample is any of the nucleic acid samples described herein, including DNA or cDNA, and may be prokaryotic, eukaryotic, or artificial in origin.
  • the NGS library is performed using methods described herein, e.g., in the section above titled “Sequence Library Construction.”
  • the performance of two different transposases may be compared by comparing the insertion bias (e.g., sequence insertion bias, e.g., G/C bias) of transposition reactions that occur when using synaptic complexes comprising the first transposase versus synaptic complexes comprising the second transposase.
  • the insertion bias e.g., sequence insertion bias, e.g., G/C bias
  • an activity fold improvements over positive control can be calculated as the percent product of transposition reactions produced by the first transposase (e.g., an engineered transposase) compared with the percent product of transposition reactions produced by the second transposase (e.g., a reference transposase), i.e., as an “FIOP Percent Product.”
  • a FIOP can be calculated as the relative calculated enzyme activity of the first transposase (e.g., an engineered transposase) compared to the second transposase (e.g., a reference transposase), such as by comparing fragmenting activity of a nucleic acid sequence (e.g., a double-stranded nucleic acid sequence) and/or transposition of
  • transposon insertion frequency is biased by the sequence of the insertion sites over the first 15 nucleotides of NGS reads putatively comprising the region of the target bound by the transposome construct.
  • bias plots can be generated from NGS data to represent this bias as a function of the distance from the insertion site.
  • overall relative bias for each transposase e.g., an engineered transposase
  • RMSD root-mean-squared deviation
  • the mean RMSD over, for example, the first 15 nucleotides (RMSD15) can be used to summarize sequence insertion bias with a single number.
  • relative target bias for each transposase can be calculated as the mean RMSD15 of a second transposase (e.g., a reference transposase) divided by the mean RMSD15 of a first transposase (e.g., an engineered transposase).
  • engineered transposases with target bias mean RMSD15 less than a reference transposase will have a ratio greater than 1 .
  • any of the above described processes for the conversion of one or more substrate compounds to product compound can further comprise one or more steps selected from: extraction; isolation; purification; and crystallization of product compound.
  • acidic compounds may exist in various salt forms that can be used interchangeably in the methods described herein. All such forms are specifically envisaged for use in the methods described herein.
  • Methods, techniques, and protocols for extracting, isolating, purifying, and/or crystallizing the product from biocatalytic reaction mixtures produced by the above disclosed processes are known to the ordinary artisan and/or accessed through routine experimentation. Additionally, illustrative methods are provided in the Examples below.
  • This plasmid construct was transformed into an E. coli strain derived from BL-21 . Directed evolution techniques were used to generate libraries of gene variants from these plasmids (See e.g., US Pat. No. 8,383,346 and WO 2010/144103, both of which are hereby incorporated by reference).
  • HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 7.1 . Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 7.1 , and analyzed on CE as described in Example 4.
  • Activity relative to SEQ ID NO: 156 was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380 produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380 observed by the reaction with SEQ ID NO: 156 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate).
  • the results are shown in Table 7.2.
  • SEQ ID NO: 302 was selected as the parent transposase enzyme.
  • Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
  • Activity relative to SEQ ID NO: 302 was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 302 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 8.2.
  • SEQ ID NO: 338 was selected as the parent transposase enzyme.
  • Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
  • Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 9.1 . Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 9.1 , and analyzed on CE as described in Example 4.
  • Activity relative to SEQ ID NO: 338 was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 338 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate).
  • Activity FIOP Activity relative to SEQ ID NO: 338
  • annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM.
  • HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 10.1 .
  • target bias relative to SEQ ID NO: 606 was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 606 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate).
  • the results are shown in Table 10.3.
  • SEQ ID NO: 972 was selected as the parent transposase enzyme.
  • Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
  • Activity relative to SEQ ID NO: 972 was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 972 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 11.2.
  • Activity relative to SEQ ID NO: 972 was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 972 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate).
  • the results are shown in Table 11.4.
  • target bias relative to SEQ ID NO: 1082 was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 1082 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate).
  • the results are shown in Table 12.3. Additional reactions were run as described above but with modified conditions. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 12.4. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 12.4 and analyzed on CE as described in Example 4.
  • target bias relative to SEQ ID NO: 1082 was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 1082 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate).
  • the results are shown in Table 12.6. Additional target bias data were collected and calculated for each target substrate/ annealed oligo pair in Table 12.3; results (unreported) were similar to those reported below.
  • Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 13.1 , and analyzed on CE as described in Example 4.
  • Activity relative to SEQ ID NO: 1580 was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 1580 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 14.2.
  • target bias relative to SEQ ID NO: 1580 was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 1580 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate).
  • the results are shown in Table 14.3. Additional target bias data were collected and calculated for each target substrate/ annealed oligo pair in Table 14.1 ; results (unreported) were similar to those reported below.
  • Activity relative to SEQ ID NO: 1580 was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 1580 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 14.8.
  • transposase variants of SEQ ID NO: 972, 1082, 1218, 1580, 1598, 1600, and 1604 were produced in shake flask and purified as described in Example 3.
  • Transposase concentrations of select variants were measured by absorption at 280 nm and performance of the normalized enzymes was measured in the preparation of libraries for next generation sequencing as follows.
  • Reactions were prepared using seqWell reagents and were evaluated on an Illumina sequencer.
  • the shake purified enzyme variants were diluted to 2.5uM in seqWell MTSB and combined in 1 :1 ratio with the seqWell i5 tagging reagents (premixed 2 barcode blend) and incubated at room temperature for 1 hour.
  • the same set of samples at concentration of 2.5uM were combined with seqWell i7 oligos in 1 :1 ratio in a separate set of reactions and incubated at room temperature for one hour.
  • the loaded i5 and i7 transposases were then combined in equal volumes and diluted to 15nM prior to library preparation.
  • active transposase variants are capable of binding target sequences, cleaving the target strand, and ligating the adapter oligonucleotides to the target fragment as shown in Figure 1 .
  • Transposon insertion frequency is biased by the sequence of the insertion sites over the first 15 nucleotides of NGS reads putatively comprising the region of the target bound by the transposome construct. Bias plots generated from NGS data represent this bias as a function of the distance from the insertion site. Overall relative bias for each variant can be calculated as the root-mean-squared deviation (RMSD) of the observed vs. expected representation of all four nucleotides at each position as shown in Scheme 1 , shown herein. Additionally, the mean RMSD over, for example, the first 15 nucleotides (RMSD15) can be used to summarize insertion bias with a single number.
  • RMSD root-mean-squared deviation
  • Relative target bias for each variant was calculated as the mean RMSD15 of SEQ ID NO: 972 divided by the mean RMSD15 of the transposase variant. Variants with target bias mean RMSD15 less than SEQ ID NO: 972 will have a ratio greater than 1 . The results are shown in Table 15.1 .
  • Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 16.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 16.1 , and analyzed on CE as described in Example 4.
  • Activity relative to SEQ ID NO: 2368 was calculated as the percent product of annealed oligos SEQ ID NO: 2375/SEQ ID NO: 2376 and SEQ ID NO: 2385/SEQ ID NO: 2386, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2375/SEQ ID NO: 2376 and SEQ ID NO: 2385/SEQ ID NO: 2386, observed by the reaction with SEQ ID NO: 2368 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 16.2.
  • target bias relative to SEQ ID NO: 2368 was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2375/SEQ ID NO: 2376 vs. product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 2368 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate).
  • the results are shown in Table 16.3. Additional target bias data were collected and calculated for each target substrate/ annealed oligo pair in Table 16.1 ; results (unreported) were similar to those reported below.
  • SEQ ID NO: 2426 was selected as the parent transposase enzyme.
  • Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
  • Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 17.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 17.1 , and analyzed on CE as described in Example 4.
  • Activity relative to SEQ ID NO: 2426 was calculated as the percent product of annealed oligos SEQ ID NO: 2375/SEQ ID NO: 2376 and SEQ ID NO: 2385/SEQ ID NO: 2386 produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2375/SEQ ID NO: 2376 and SEQ ID NO: 2385/SEQ ID NO: 2386, observed by the reaction with SEQ ID NO: 2426 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 17.2.
  • SEQ ID NO: 2540 was selected as the parent transposase enzyme.
  • Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
  • Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 18.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 18.1 , and analyzed on CE as described in Example 4.
  • Activity relative to SEQ ID NO: 2540 was calculated as the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, observed by the reaction with SEQ ID NO: 2540 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate).
  • the results are shown in Table 18.2.
  • SEQ ID NO: 2540 was selected as the parent transposase enzyme.
  • Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
  • Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 19.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 19.1 , and analyzed on CE as described in Example 4.
  • Activity relative to SEQ ID NO: 2540 was calculated as the percent product of annealed oligos SEQ ID NO: 2369/SEQ ID NO: 2370, SEQ ID NO: 2371/SEQ ID NO: 2372, SEQ ID NO: 2373/SEQ ID NO: 2374, SEQ ID NO: 2375/SEQ ID NO: 2376, SEQ ID NO: 2377/SEQ ID NO: 2378, SEQ ID NO: 2379/SEQ ID NO: 2380, SEQ ID NO: 2381/SEQ ID NO: 2382, SEQ ID NO: 2383/SEQ ID NO: 2384, and SEQ ID NO: 2385/SEQ ID NO: 2386, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2369/SEQ ID NO: 2370, SEQ ID NO: 2371/SEQ ID NO: 2372, SEQ ID NO: 2373/SEQ ID NO: 2374, SEQ ID NO: 2375/SEQ ID
  • target bias relative to SEQ ID NO: 2540 was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 2540 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate).
  • the results are shown in Table 19.3. Additional target bias data were collected and calculated for each target substrate/ annealed oligo pair in Table 19.1 ; results (unreported) were similar to those reported below.
  • target bias relative to SEQ ID NO: 3402 was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 3402 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate).
  • the results are shown in Table 20.3. Additional target bias data were collected and calculated for each target substrate/ annealed oligo pair in Table 20.1 ; results (unreported) were similar to those reported below.
  • Activity relative to SEQ ID NO: 3788 was calculated as the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698 produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698 observed by the reaction with SEQ ID NO: 3788 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 22.2.
  • Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 45 mins. After 45 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 24.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 24.1 , and analyzed on CE as described in Example 4.
  • Activity relative to SEQ ID NO: 4278 was calculated as the percent product of annealed oligos SEQ ID NO: 5695/SEQ ID NO: 5696, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5695/SEQ ID NO: 5696, observed by the reaction with SEQ ID NO: 4278 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 24.2.
  • SEQ ID NO: 4278 was selected as the parent transposase enzyme.
  • Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2. Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 45 mins.
  • annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM.
  • HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 25.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 25.1 , and analyzed on CE as described in Example 4.
  • SEQ ID NO: 4642 was selected as the parent transposase enzyme.
  • Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
  • Activity relative to SEQ ID NO: 4642 was calculated as the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, observed by the reaction with SEQ ID NO: 4642 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate).
  • the results are shown in Table 26.2.
  • SEQ ID NO: 4642 was selected as the parent transposase enzyme.
  • Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2. Reactions were performed in 96-well format BioRad PCR plates. 100 nL of 100% HTP lysate variants in lysis buffer (Table 27.1) were combined in 1 :1 volume ratio with 2.5 pM seqWell i5 tagging reagents and incubated at room temperature for 1 hour with mild shaking.
  • Relative target bias for each variant was calculated as the mean RMSD15 of SEQ ID NO: 4642 divided by the mean RMSD15 of the transposase variant. Variants with target bias mean RMSD15 less than SEQ ID NO: 4642 will have a ratio greater than 1 . The results are shown in Table 27.2.
  • Activity relative to SEQ ID NO: 4984 was calculated as the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, observed by the reaction with SEQ ID NO: 4984 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 28.2.
  • SEQ ID NO: 5412 was selected as the parent transposase enzyme.
  • Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
  • SEQ ID NO: 5422 was selected as the parent transposase enzyme.
  • Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
  • SEQ ID NO: 972 was selected as the parent transposase enzyme.
  • Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
  • Relative target bias for each variant was calculated as the mean RMSD15 of SEQ ID NO: 972 divided by the mean RMSD15 of the transposase variant. Variants with target bias mean RMSD15 less than SEQ ID NO: 972 will have a ratio greater than 1 .
  • the results are shown in Table 35.1 .

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Abstract

The present invention provides engineered transposase polypeptides useful in preparation of next generation sequencing libraries, as well as compositions, methods of utilizing these engineered polypeptides, and polynucleotides encoding the engineered transposases. The present application further relates to methods for using engineered transposases, e.g., in the preparation of nucleic acid libraries for nucleic acid sequencing. More specifically, the invention pertains to methods that use engineered transposases having improved performance to improve the transposition reactions during the preparation of nucleic acid libraries.

Description

ENGINEERED TRANSPOSASES AND METHODS OF USE THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 63/622,732, filed January 19, 2024, and U.S. Provisional Application No. 63/623,064, filed January 19, 2024, both of which are herein incorporated by reference, in their entireties.
REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 17, 2025, is named “51178-017WO2_Sequence_Listing_1_17_25”, and is 12,833,483 bytes in size.
TECHNICAL FIELD
The present invention provides engineered transposase polypeptides useful in the preparation of next generation sequencing (NGS) libraries, as well as compositions and methods of utilizing these engineered polypeptides.
BACKGROUND
Transposases are enzymes known to facilitate the transposition of fragments of DNA (transposons) from a donor strand to a target strand at sites flanked by short inverted-repeat sequences or end sequences (ESs). The retroviral integrase superfamily of proteins includes a number of transposases, including Tn5, an IS4 transposase. The transposition mechanism is initiated by the binding of two transposases to donor DNA ESs to form the dimeric synaptic complex or transposome, which catalyzes cleavage of donor DNA. The transposome then transfers the transposon to the target strand (Gradman, R.J., & Reznikoff, W.S. (2008). Tn 5 Synaptic Complex Formation : Role of Transposase Residue W 450.). Two divalent metal ions (Mg2+ or Mn2+) bind to the active site and are critical for catalysis (Lovell, S., Goryshin, L, Reznikoff, W. et al. Two-metal active site binding of a Tn 5 transposase synaptic complex. Nat. Struct. Mol. Biol. 9:278-281 (2002).)
In nature, transposases have limited activity. However, several mutations in Tn5 are known to enhance transposase activity, including L372P, E54K, E110K, E345K, and P242A/G (Reznikoff, W.S. (2003), Tn5 as a model for understanding DNA transposition. Molecular Microbiology, 47: 1199-1206.) Additionally, a 19 bp ES known as mosaic end (MEs) is associated with hyperactivity in Tn5 transposases.
Next generation sequencing (NGS) has facilitated sequencing of large DNA libraries using limited samples. However, sample preparation is time-consuming and resource-intensive, necessitating development of more efficient protocols. Tn5 transposase has been used to reduce costs and improve efficiency in preparing samples for large-scale sequencing projects (Hennig BP, Veiten L, Racks I, Tu CS, Thoms M, Rybin V, Besir H, Remans K, Steinmetz LM. Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol. G3 (Bethesda). 2018 Jan 4;8(1):79-89.) In one example, a hyperactive Tn5 with L372P and E54K mutations was used with MEs in a tagmentation protocol to simultaneously fragment sample DNA and bind sequencing adapters for NGS library preparation (Picelli S, Bjorklund AK, Reinius B, Sagasser S, Winberg G, Sandberg R. Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Res. 2014 Dec;24(12):2033-40.)
Improvements to these methods have been reported (Hennig et. al)., and various commercial sample preparation methods now incorporate Tn5 transposase tagmentation. In one instance, a Tn5 transposase is used to create normalized libraries suitable for NGS without the need for independent adjustment or parallelized normalization of input samples or resulting libraries (PCT/US2016/013753).
However, existing transposase NGS prep methods are limited primarily by their high cost. A transposase with increased DNA fragmentation or cleavage and increased adapter loading or ligation would expand the utility of existing transposase NGS library preparation methods. Transposases with other improved properties, including increased thermostability, increased activity at elevated temperatures, increased soluble expression, decreased inhibition, increased binding to a target polynucleotide or adapter polynucleotide, and/or decreased sequence insertion bias as compared to another wild-type or engineered transposase, are also highly desirable.
SUMMARY
The present invention provides engineered transposase polypeptides useful in preparation of next generation sequencing libraries, as well as compositions and methods of utilizing these engineered polypeptides. The transposases of the present invention are derived from a wild-type (WT) polypeptide described by UniProt accession number A0A3L8PTF2 a predicted IS4 family transposase from Parashewanella curva or from a modified hyperactive Tn5 transposase that contains E54K and L372P amino acid substitutions and is described by NCBI Protein accession number HQ908071 .1 . A C- terminal 6-histidine tagged version of the transposases (SEQ ID NO: 2 or SEQ ID NO: 5704, respectively) recognize the 19 bp Tn5 mosaic end (ME) transposon donor recognition sequence. Beneficial properties of these engineered transposases include increased binding to adapters or donor DNA to form a transposome, increased DNA cleavage, increased DNA fragmentation, increased activity at elevated temperatures, increased soluble expression, increased ligation of the adapters or donor DNA to the target DNA, decreased inhibition and/or decreased sequence insertion bias, as compared to a wild-type transposase or a reference transposase.
In some embodiments, the present invention provides an engineered transposase polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, optionally comprising at least one substitution or one substitution set at one or more positions, wherein the positions are numbered with reference to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, and wherein the engineered transposase polypeptide has increased polynucleotide fragmentation or cleavage activity, increased thermostability, increased activity at elevated temperatures, increased soluble expression, decreased inhibition, increased adapter loading or adapter ligation (i.e. tagging), increased binding to a target polynucleotide, and/or decreased sequence insertion bias as compared to another wild-type transposase or engineered transposase. These engineered transposase polypeptides with one or more amino acid substitutions or substitution sets are described, below, in the detailed description of the invention.
In some embodiments, the present invention provides engineered transposases comprising a polypeptide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, or a functional fragment thereof, wherein the engineered transposase comprises at least one substitution or substitution set in its polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704.
In some additional embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 10, 13, 24, 35, 39, 44, 46, 51 , 54, 55, 62/361 , 72, 75, 82, 89, 92, 107, 128, 137, 164, 167, 177/359, 191 , 192, 193, 194, 201 , 217, 281 , 289, 291 , 291/340, 305, 309, 310, 317, 331 , 337, 340, 346, 352, 353, 357, 358, 359, 362, 371 , 396, 414/435, 415, 428, 437, 443, 447, and 448, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 4, 5, 6, 8, 10, 10/317, 10/317/358, 10/317/359, 10/346, 10/346/359, 10/358, 10/359, 12, 12/373, 13, 15, 16, 18, 19, 26, 30, 30/154, 31 , 34, 35, 37, 38, 68, 70, 73, 74, 93, 106, 118, 126, 134, 135, 143, 146, 154/453, 163, 182, 207/286, 208, 226, 237, 246, 247, 259, 262, 263, 264, 280, 282, 286, 287, 288, 289, 291 , 292, 293, 298, 305, 309, 310, 313, 317, 317/346, 317/359, 317/445, 320, 346, 351 , 355, 356, 358, 359, 360, 368, 369, 410, 411 , 422, 426, 427, 428, 446, 448, 453, and 454, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 18/73/74/259, 18/73/286/289/411 , 18/74/118/259/286/289/411 , 18/74/259, 18/74/259/286/289, 18/74/259/286/411 , 18/118/259, 18/259/286/289/411 , 18/259/289/411 , 18/289/411 , 73/74/259/286/289, 73/74/259/289, 73/259/286, 74/259, 74/259/289, 74/286/289/411 , 259, 259/286/289, and 289, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 156.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 16/19/355/427, 16/37/146/286/287/289/310/313/355, 16/146/226/287/289/355, 19/37/146/286/287/310/355, 19/135/286/287/289/355, 19/182/355, 37/70/226/310/313/355, 37/74/310/355/427, 37/135/146/182/226/286/287/355/368, 37/146/226/289/427, 37/146/287/289/427, 38, 41 , 42, 43, 44, 45, 47, 48, 50, 53, 54, 55, 70/74/226/286/287/289/368/427, 70/74/355/368, 70/135/146/150/182/286/289/313/427, 74/310/313/355, 74/310/355, 74/355/427, 101 , 103, 117, 135/226/286/289/355/368, 135/355, 146, 146/150/287/289/310/355, 146/182/355, 146/226/286/355, 146/286/287/289/355/427, 146/289, 146/427, 150/226/310/313, 182/226/355, 182/355, 244, 247, 249, 251 , 287/355/427, 300, 317/411 , 355, 393, 397, 398, 399, 401 , 405, 407, 408, 409, 411 , 413, 426, 429, 431 , 439, 444, 446, 450, 453, 454, 457, 459, 460, 464, 466, 467, 469, 470, 471 , 472, 474, 475, 476, and 478, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 302.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 16, 16/313, 16/409/411 /427, 37/150/408, 37/313/317/408/411 , 37/317/405/408, 37/317/405/426, 37/405/409/427, 150, 150/313/405/411 , 150/405/406, 150/408, 150/408/411 , 226/405, 317/460, and 408/426, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 338.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 12, 13, 18, 19, 22, 26, 31 /262, 34, 60, 79, 80, 81 , 82, 92, 106, 107, 113, 115, 132, 134, 136,
140, 143, 152, 160, 163, 167, 168, 175, 179, 183, 195, 199, 201 , 202, 215, 216, 218, 220, 223, 226, 233,
235, 236, 237, 243, 255, 259, 261 , 262, 266, 268, 270, 273, 282, 290, 292, 293, 309, 319, 320, 326, 343,
346, 365, 370, 374, 380, 382, 386, 389, 390, 408, 412, 414, 415, 424, and 442, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 606.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 2, 12, 13, 15, 16, 18, 19, 22, 26, 30, 31/262, 34, 51 , 60, 69, 79, 82, 84, 87, 88, 106, 107, 113, 114, 115, 128, 132, 134, 135, 136, 140, 143, 146, 150, 152, 160, 163, 167, 168, 175, 179, 183, 195,
198, 199, 201 , 215, 217, 218, 219, 220, 223, 224, 226, 227, 231 , 233, 235, 236, 243, 254, 255, 259, 261 ,
266, 267, 268, 270, 272, 273, 275, 277, 279, 282, 287, 289, 290, 291 , 292, 293, 304, 307, 309, 319, 320,
343, 344, 346, 355, 365, 370, 373, 380, 382, 386, 389, 390, 408, 412, 414, 415, 418, 423, 424, and 468, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 606.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 44/45/47/408/453, 44/45/47/453, 44/45/47/453/454, 44/45/103/453/457, 44/45/453/454/457, 44/47/103/393/408/453/454, 44/47/103/453/454/457, 44/47/393/408/453/457, 44/47/393/453/457, 44/103/408/453/454, 45/47/103/393/453/454/457, 45/47/393/453/454, 45/47/408/423/453/457, 45/47/453, 45/103/393/453/454, 45/453/454, 47/103/408/453/454, 47/393/408/453/454/457, 47/393/453/454, 47/408/453/457, 53/54/244/450, 103/393/408/453/457, 244/450, 408/453/454, 446/450/464, and 453/457, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 606. In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 38/45/53, 38/45/53/54/476, 38/45/53/244, 38/45/53/470/472, 38/45/54/244, 38/45/54/470/474/478, 38/45/472/476/478, 38/45/476, 38/53/54, 38/53/54/244, 38/53/54/244/472/478, 38/53/472, 38/54/244/470/474/476/478, 38/244/470/472/474/478, 38/478, 45/53/54/244/470/474/476/478, 45/53/54/470/474/476, 45/53/54/472/478, 45/54/244/474/478, 53, 53/54/470/476/478, 53/244/474/478, 54, 54/244, 54/244/472/476, 54/472, 244, and 474/478, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 22/38/45/107/259/317/412, 22/38/45/259/317/412, 22/107, 22/107/317/412, 22/134, 22/134/220, 22/134/220/317, 22/134/317/412, 22/317, 38, 38/45/81/143/244, 38/53/243, 38/81 , 38/81/244, 38/107/134/220, 38/132, 38/220/317, 45/81 , 45/81/243, 45/81/243/343, 45/143/243/270/343, 45/244/270, 53/115/163/243/270/343, 53/143/243/270/343, 53/270, 64/134/220/317/412, 81/132/243, 107, 107/134/317, 115/244/270, 134/259, 143/243/270, 243, 243/270, 244, 244/270, 259, 270, and 317, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 22/38/45/107/259/317/412, 22/38/45/259/317/412, 22/38/64/134/220, 22/45/220/259, 22/64/107/259/317/412, 22/64/134/220, 22/107, 22/134, 22/134/317, 22/134/317/412, 22/220, 22/220/412/415, 22/259/317, 22/259/317/412, 22/317, 38, 38/45/81/143/244, 38/45/143/270, 38/53/243, 38/81 , 38/81/143/243/270, 38/81/183/243/270, 38/81/243, 38/81/244, 38/107/134/220, 38/132, 38/220/317, 38/270/343, 45/81 , 45/81/132/243/270, 45/81/243, 45/143/243/270/343, 45/244/270, 53/143/243/270/343, 53/244/270, 53/270, 64, 64/107/134, 64/107/317, 64/134, 64/134/220/317/412, 81/132, 81/132/243, 107, 107/134/317, 107/412, 115/244/270, 143/243/270, 220/259, 243, 243/270, 244/270, 255, 259, and 270, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 143, 143/175/272/273/415, 152/168/216/237, 152/168/423, 160, 160/201/272, 160/201/273/415, 160/226, 160/244/272/273/415, 160/415, 168, 168/187/216/255/423, 168/216/237, 168/255/423, 168/290/423, 187, 187/423, 199/201/226, 199/201/415, 201 , 216, 216/423, 226/244/415, 226/415, 237, 237/290, 244, 255, 272, 290, 290/423, 415, and 423, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1082.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 143, 143/160/226/415, 143/175/272/273/415, 152/168/216/237, 152/168/423, 160, 160/201/272, 160/201/273/415, 160/226, 160/244/272/273/415, 160/415, 168, 168/187/216/255/423, 168/216/237, 168/216/237/255/343, 168/255/423, 168/290/423, 187, 199/201/226, 199/201/415, 201 , 216, 216/423, 226/244/415, 226/415, 237, 237/290, 244, 255, 255/343, 272, 290, 290/423, 415, and 423, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1082.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 72, 90, 91 , 92, 93, 95, 122, 129, 141 , 142, 143, 144, 145, 150, 151 , 151/155, 152, 154, 155, 157, 160, 161 , 161/313, 164, 165, 168, 180, 181 , 184, 193, 197, 204, 205, 205/472, 206, 299, and 320, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1082.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 90, 92, 122, 129, 141 , 143, 144, 145, 150, 151 , 151/155, 152, 154, 155, 155/265, 157, 160, 161 , 164, 165, 168, 180, 181 , 184, 193, 197, 204, 205, 205/472, 206, 299, and 320, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1082.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 129, 129/160, 129/160/164, 129/160/184, 129/164, 129/164/184, 129/205, 146/152/244, 150/155/226, 150/155/244, 152, 154, 154/160/184, 154/164, 154/205, 155, 160, 160/164, 160/164/184, 160/184, 164, 164/204, 164/205, 204, 205, 226, 226/244, and 244, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1218.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 129, 129/143, 129/154/164, 129/154/164/205, 129/154/205, 129/160, 129/160/164, 129/160/205, 129/164/205, 143, 143/150/155/226/244, 143/150/155/244, 143/150/226, 143/150/226/244/343, 143/150/226/343, 143/150/244, 143/150/343, 143/152, 143/152/226, 143/152/226/244, 143/152/226/343, 143/152/343, 143/155, 143/155/226/244, 143/155/226/244/343, 143/155/343, 143/226, 143/226/244/343, 143/226/343, 143/244, 143/244/343, 143/343, 146/152/244, 150/155/226, 150/155/244, 150/155/244/343, 150/226/244/343, 152, 152/174/244/343, 152/226, 152/226/343, 152/343, 154, 154/160/184, 154/164, 154/164/204, 154/164/204/205, 154/205, 155, 160, 160/164, 160/164/184, 164, 205, 226, 226/244, and 244, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1218.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 98/129/157/168, 129/157/168, 151 , 151/152/255, 151/152/273, 151/255, 151/343, 157, 157/164, 157/164/168, 157/168, 164, 164/255, 168, 183, 255, 255/343, and 343, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 129, 129/157, 129/157/168, 129/164, 129/164/168/183, 129/164/183, 143, 143/151 , 143/151/152, 143/151/164, 143/164, 143/164/255, 143/164/343, 143/255, 143/343, 151 , 151/152, 151/152/164/343, 151/152/343, 151/255, 151/255/343, 151/343, 152, 152/255, 157, 157/164/168, 157/168, 164, 164/168, 164/255, 164/447, 168, 183, 255, 255/343, 298/343, and 343, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 69, 69/164, 72, 73, 91 , 92/164, 95, 96, 97, 98, 110, 112, 113, 121 , 121/164, 122, 122/164, 124, 126, 127, 139, 140, 141 , 144, 144/164, 145, 146, 147, 148, 150, 150/164, 151 , 151/164, 153, 153/164, 155, 156, 157, 159, 160, 161 , 163, 164, 164/172, 164/173, 164/182, 164/188, 164/191 , 164/192, 164/195, 164/197, 164/199, 164/202, 164/205/249, 164/209, 165, 166, 167, 169, 170, 171 , 172, 173, 177, 184, 187, 188, 190, 191 , 192, 193, 194, 195, 197, 197/266, 198, 199, 200, 202, 203, 204, 205, 208, 209, 210, 321 , 324, 331 , and 349, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 69, 69/164, 72, 73, 91 , 92/164, 95, 97, 98, 121/164, 126, 127, 139, 140, 142, 144, 144/164, 145, 146, 146/164, 148, 150, 150/164, 151 , 153, 153/164, 155, 156, 157, 160, 163, 164/173, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/205/249, 164/209, 165, 166, 167, 169, 171 , 172, 173, 177, 182, 187, 188, 190, 191 , 192, 194, 195, 197/266, 198, 199, 200, 202, 203, 204, 205, 208, 209, 210, 324, 331 , and 349, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 73, 91 , 93, 95, 97, 98, 110, 112, 122, 122/164, 124, 127, 139, 144, 145, 146, 146/164, 147, 148, 150, 150/164, 151 , 151/164, 153, 153/164, 155, 156, 157, 159, 160, 161 , 162, 163, 164, 164/182, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/205/249, 164/209, 165, 166, 167, 169, 171 , 172, 177, 184, 187, 188, 190, 191 , 192, 193, 195, 197/266, 198, 199, 200, 204, 205, 206, 207, 208, 209, 210, and 324, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 69, 73, 91 , 127, 140, 144, 145, 146, 146/164, 150, 150/164, 151 , 153, 155, 156, 157, 160, 163, 164, 164/172, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/209, 165, 167, 169, 171 , 172, 182, 183, 188, 191 , 192, 193, 195, 196, 198, 199, 200, 204, 205, 208, 210, and 324, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 38/143/168/220/290/317/423, 38/152/154/164/168/220/290/317/423, 38/152/168/220/290/317/343/423, 38/152/168/220/290/317/423, 38/154/164/168/220/290/317/423, 38/168/220/290/317/423, and 38/220/317, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 73/143/169/191/193, 73/156, 73/156/192/435, 73/156/210, 73/163, 73/163/210, 73/192/193, 127, 127/160/172/198, 127/172, 127/198, 146, 146/151/188, 146/324, 151/171/324, 151/324, 156/169, 156/192, 157/171 , 163, 163/169, 163/169/191/192/193, 165/171/324, 165/188/324, 165/324, 169, 171/188/324, 171/324, 172, 188, 192, 192/193, 198, 210, and 324 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2368.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 73, 73/163/191/192/193, 127/160/172, 127/160/172/198, 144, 160, 160/172, 163/191/192, and 192 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2368. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 2396. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 2396, wherein the C-terminal histidine tag is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 2396, wherein the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof is replaced or removed.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 8, 13, 17, 18, 20/146/157/167, 26, 34/38, 38/39/45, 67, 70, 73, 73/150/155/169, 73/150/171/192, 73/155, 73/155/169/199, 73/155/171 , 73/192, 73/199, 78, 82, 84, 86, 86/384, 89, 90, 91 , 95, 102, 110, 111 , 113, 114, 115, 122/127, 126/127, 127/131/165, 127/133, 143, 143/146, 143/146/153, 143/146/157, 143/146/167, 143/146/167/188, 143/146/188, 143/153/167/188/246, 143/156/157/167/195, 143/156/188, 143/188, 143/188/195, 143/195, 144, 145, 145/151 , 146, 146/152, 146/153/157, 146/153/157/167, 146/153/167, 146/157, 146/157/167, 146/167, 150, 150/155/169, 150/155/169/192, 150/155/192, 150/169, 150/192, 150/199, 151/152/154, 152/154, 152/154/155/160, 153/157/167, 155/171 , 155/192, 156/270, 160/371 , 167/188, 168/172, 169, 184, 190, 192/198, 198, 206, 208, 209, 210, 214/220, 215/220, 216/220, 217/220, 219/220, 220, 220/222, 220/225, 220/226, 231 , 235, 242, 257, 259, 261 , 262, 267, 268, 269, 270, 272, 274, 275, 276, 277, 278, 282, 283/290, 284/290, 287/290, 288/290, 289/290, 290, 290/291 , 290/292, 290/293, 290/294, 304, 305, 305/373, 306, 307, 309, 317, 329, 333, 334, 339, 341 , 344, 345, 346, 352, 353, 355, 358, 366, 368, 370, 372, 373, 375, 376, 378, 379, 380, 381 , 382, 383, 384, 385, 386/393, 387/393, 389/393, 390/393, 391/393, 392/393, 393/394, 414, 415, 420/423, 422/423, 423, 423/425, 432, 440, 441 , 443, 469, and 472 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2426.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 8, 18, 25, 29, 30, 67, 73, 73/150/155/169, 73/150/155/171 , 73/150/171 /192, 73/171 , 73/192, 73/199, 74, 82, 86, 86/384, 113, 115, 118, 127/128, 143, 150, 150/155/169, 150/155/169/192, 150/155/171 , 150/155/171/199, 150/155/192, 150/155/199, 150/192, 150/199, 155/192, 168/171/172, 195/198, 197/198, 198/201 , 198/203, 198/205, 235, 252, 256, 257, 262, 263, 267/343, 268, 269, 270, 272, 273, 276, 277, 288/290, 289/290, 290, 290/291 , 290/292, 290/294, 298, 304, 305, 307, 333, 346, 355, 366, 370, 386/393, 389/393, 393/395, 440, and 472 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2426.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 66, 67, 70, 101 , 102, 103, 104, 106, 107, 108, 113, 114, 115, 117, 118, 143, 152, 154, 235, 236/251 , 237, 238, 242, 243, 246, 247, 249, 250, 251 , 256/352, 257, 259, 285, 286, 287, 288, 289, 290, 292, 293, 294, 295, 296, 322, 323, 326, 329, 330, 333, 339, 340, 343, 344, 345, and 352. and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2540.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 18, 18/113/270, 18/113/270/329/370, 18/113/270/370/384, 18/113/270/370/412, 18/113/329/384, 18/259/370/384, 18/270, 18/270/370, 18/270/370/384, 18/370, 18/370/384, 73, 73/115, 73/115/184/333, 73/115/184/341 , 73/115/256, 73/184/256, 73/184/273/333, 73/184/333, 73/256, 73/256/375, 73/273, 73/273/333/375, 73/273/375, 73/333, 73/333/375, 73/375, 73/379, 77/233/238/376, 77/233/334/376, 77/262/376, 77/276, 77/334/376, 82, 82/127, 82/127/144/235/290, 82/127/144/266, 82/127/144/266/290, 82/127/144/378, 82/127/235, 82/127/290, 82/144/266/290/378, 82/144/266/378, 82/235/266, 82/235/266/378/383, 82/235/290, 82/235/378, 82/266, 82/266/378, 82/378, 113/270/370/384, 115/256/333/375, 115/256/375, 115/273/375, 115/375, 127/144/290, 127/149/150, 127/266/290/378, 127/378/383, 144/235/290, 144/266/290, 233/334, 233/376, 235, 235/290, 256/333, 262, 266/378, 270/370, 273, 276/352, 290, 290/378, 333, 358, 370, 375, and 378 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2540.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 18, 18/113, 18/113/252, 18/113/252/336/384, 18/113/252/370, 18/113/270, 18/113/270/370/384, 18/259/370/384, 18/270, 18/270/370, 18/270/370/384, 18/370, 18/370/384, 73, 73/115/184/341 , 73/115/341 , 73/131/256/273/333/341 /375, 73/184/256, 73/184/256/341 , 73/184/256/375, 73/184/273/333, 73/184/333, 73/184/341/375, 73/256, 73/256/273, 73/256/273/375, 73/256/333/375, 73/256/375, 73/273, 73/273/333/341 , 73/273/333/375, 73/273/375, 73/333, 73/333/341/375, 73/333/375, 73/341 , 73/375, 73/379, 77, 77/118, 77/118/143/262/276/334/376, 77/118/143/334/352/376, 77/118/143/376, 77/118/233, 77/118/233/352/376, 77/118/262/334/376, 77/118/334, 77/143/262/276, 77/143/262/334, 77/143/376, 77/233/238/376, 77/233/262, 77/233/276/376, 77/233/334/376, 77/262/376, 77/276, 77/334/376, 77/352, 82, 82/127, 82/127/144/235/290, 82/127/144/266, 82/127/144/266/290, 82/127/144/378, 82/127/235, 82/127/235/266/358, 82/127/290, 82/127/290/358, 82/127/290/378, 82/144/266/290/378, 82/144/266/378, 82/150/235/266, 82/235/266, 82/235/266/290, 82/235/266/378/383, 82/235/290, 82/235/378, 82/266, 82/266/378, 82/290, 82/378, 113, 113/252, 113/252/270/370, 113/270/370/384, 113/336, 118/233/276/334, 118/334, 127/235/266, 127/266/290/378, 127/378/383, 143, 143/276, 143/276/334, 144/235/290, 144/266/290, 144/290/358, 233/334, 233/376, 235, 235/266/290/358/383, 235/358, 252/329/336/370, 252/384, 256/333, 256/375, 262, 262/276, 262/334, 266/290, 266/378, 270/370, 273, 276/352, 290, 290/378, 333, 334, 334/376, 336/370, 341 , 358, 370, 375, and 378 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2540.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 18, 18/77/118/169/233/238/256/266/270, 18/77/150/256/376, 18/118, 18/118/143/169/238/256/266/333, 18/118/150/169/184/238/256/270/378, 18/118/169, 18/118/233/238, 18/143, 18/143/256, 18/150/169, 18/150/266, 18/169/184/290/333/376, 18/169/233/333, 18/169/392, 18/184/270, 18/233/256/334, 18/256/266/290/378, 77/113/118/256/266/290, 77/118/290, 77/169/270/290, 77/233, 77/233/256, 77/238, 113/118/169/184/256/376, 113/169, 113/233, 118, 118/143/238, 118/233/238/256/378, 118/233/238/270, 143/169/233/270, 143/233/266/290/376, 143/238/270/290/378, 143/256/334, 143/290, 169, 169/256/266, 183/270/333, 233/238, 233/238/376, 233/392, 238/256, 256/290/303, 270, 290, 376, and 378 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3402.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 18, 18/77/118/169/266/334/376, 18/77/150/256/376, 18/77/256/290/334, 18/113/238, 18/118, 18/118/143/169/238/256/266/333, 18/118/143/169/376, 18/118/169, 18/118/233/238, 18/118/266, 18/143, 18/143/169/233/238/358, 18/143/233/238/290/376, 18/143/256, 18/143/290, 18/143/333/334/378, 18/150/169, 18/150/266, 18/169/184/290/333/376, 18/169/233/333, 18/169/238/270/334/376, 18/169/392, 18/233/256/334, 18/233/266/290/376, 18/256/266/290/378, 18/266/270/378, 18/378, 77/113/121/143/233/334, 77/118/169/233/266/270, 77/118/238/376, 77/118/290, 77/169/270/290, 77/233, 77/238, 113/238/376, 118, 118/143/238, 118/143/392, 118/169/184/233/266/270/333, 118/233/238/256/378, 118/256/290/333/334/376/378, 118/256/334/376, 118/266/290, 143/169/233/270, 143/233/266/290/376, 143/238/270/290/378, 143/256/334, 143/290, 143/364, 150/233/333, 169, 169/256/266, 183/270/333, 233/238, 233/238/376, 233/392, 238/256, 238/266/270/378, 256/266/334, 266/270/376, 266/333, and 378 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3402.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 77/156, 77/156/270, 77/184/244, 77/184/333, 77/244/270, 77/270, 143, 143/169/238/257/344, 143/169/255, 143/169/255/257, 143/169/343, 143/255, 143/255/257, 143/255/344, 143/257, 156, 156/270, 169/238, 169/238/255/257/344, 169/257/344, 169/344, 184, 184/244/324, 238, 238/255/344, 244/270, 257/344, and 270 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3662.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 77, 77/156/184/244/324, 77/184/270, 143, 143/238, 143/238/255, 143/238/255/343, 143/238/255/392, 143/238/344, 143/255, 143/257/392, 143/344, 169, 169/238/255/344, 169/238/344, 169/255, 169/343, 169/392, 238/344, 255, 255/257, 255/257/392, 257, and 344 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3662.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 20, 22, 26, 42, 43, 44, 45, 50, 51 , 52, 53, 54, 55, 60, 62, 64, 65, 95, 97, 99, 103, 104, 111 , 112, 115, 115/143/169, 115/169/343, 115/343, 116, 116/343, 143, 143/169, 154/439, 169, 169/324, 169/324/343, 169/343, 186, 234, 236, 238, 238/246, 239, 239/244, 241 , 242, 243, 244, 250, 251 , 254, 255, 260, 283, 284, 295, 296, 298/326, 319, 322, 323, 324, 324/343, 326, 328, 329, 333, 334, 338, 340, 341 , 343, 344, 347, 366, 434, 439, 442, 443, 444, 445, 446, and 447 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3788.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 26, 26/236, 26/236/260/333, 26/236/295, 26/260, 26/260/295, 26/260/295/333, 26/295/319, 26/333, 64, 64/244, 64/251/260/347, 64/260/295/333, 64/260/347, 103/115/255, 103/115/343, 103/115/343/344, 103/143/255/284/343/344, 103/169/343/344, 103/255/284, 103/343/344, 115, 115/143/255, 115/143/255/343, 115/143/284, 115/143/343, 115/143/344, 115/169, 115/169/343, 115/255, 115/255/344, 115/284, 115/284/343/344, 115/284/344, 115/343, 115/343/344, 115/344, 143, 143/169/255/284/343/344, 143/169/343/344, 143/255, 143/255/284/343/344, 143/255/343, 143/255/343/344, 143/255/344, 143/343/344, 169, 169/255, 169/255/284, 169/255/343, 169/284, 169/343, 169/344, 236, 236/244/333, 236/260, 236/347, 244, 244/251 , 244/251/319, 244/260, 244/295, 244/347, 251 , 251/260/295/347, 251/333, 255, 255/284, 255/343, 255/343/344, 255/344, 259, 260, 260/295, 260/333, 284, 284/343/344, 284/344, 295, 295/333, 333, 343, 343/344, 344, and 347 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4150.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 115, 115/117, 115/117/118, 115/117/118/152, 115/117/118/169, 115/117/118/246, 115/117/152, 115/117/152/169, 115/117/152/169/246, 115/117/152/246, 115/117/169, 115/117/246, 115/118, 115/118/152, 115/118/152/169, 115/118/152/169/246, 115/118/169, 115/118/246, 115/152, 115/152/169, 115/152/169/246, 115/152/246, 115/152/246/374, 115/169, 115/169/246, 115/246, 117, 117/118, 117/118/152, 117/118/152/169, 117/118/152/246, 117/118/169, 117/118/246, 117/152, 117/152/169, 117/152/246/288, 117/169, 117/246, 118/246, 152, 152/169, 152/169/246, 152/246, 169, 169/246, and 246 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4278. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4642. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4642, wherein the C-terminal histidine tag is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4642, wherein the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof is replaced or removed. In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 73, 101 , 114, 115, 118, 143, 145, 150, 151 , 152, 153, 154, 156, 157, 160, 161 , 163, 166, 167, 168, 169, 171 , 172, 190, 191 , 192, 195, 213, 214, 218, 219, 223, 225, 233, 233/343, 234, 235, 236, 237, 238, 239, 239/250, 243, 244, 249, 251 , 254, 255, 257, 258, 259, 261 , 262, 262/283, 277/292, 283, 284, 286, 289, 292, 295, and 296 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4278.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 93, 113, 113/114, 113/115, 113/143, 115/118, 115/177, 118/143/163/168/195/255/315, 118/143/255, 118/163/168/214/255/315, 118/168/214/315, 118/255, 124, 126, 129, 134, 143/154/255, 143/156/168/216/255, 143/163, 143/163/255, 143/168, 143/168/169/255/315, 143/255, 148, 156/214/315, 159, 163, 163/168, 163/168/169, 163/168/169/255/315, 163/168/169/315, 163/168/195/214/255/315, 163/214, 163/255, 163/255/315, 168/315, 186, 194, 197, 206, 207, 208, 210, 213, 255/315, 277, 293, 294, 315, 317, 320, 322, 328, 329, 330, 333, 352, 366, 392, and 445 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4642. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4872. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4872, wherein the C-terminal histidine tag is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4872, wherein the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4954. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4954, wherein the C-terminal histidine tag is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4954, wherein the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof is replaced or removed.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 127, 128, 129, 140, 180, 182, 194, 277, 293, 294, 299, 300, 317, 319, 320, 324, 333, 356, 366, and 392 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4642.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 113, 117, 118, 151 , 191 , 196, and 205 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4984.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 114/115/143, 114/115/143/233, 114/115/233, 114/115/343, 114/143, 114/143/151/233/238/261 , 114/143/213/261/343, 114/143/233, 114/143/233/238, 114/151/261/343, 114/213/238, 114/343, 115, 115/143, 115/143/151/154/213/261 , 115/143/151/154/233/343, 115/143/151/233/261 , 115/143/151/233/343, 115/143/154/213/233/238/261 , 115/143/213, 115/143/233, 115/143/343, 115/151/154/233, 115/151/261 , 115/213/233, 115/213/233/238, 115/213/261 , 115/233/238, 115/238, 115/343, 115/348, 143, 143/151/154, 143/151/154/213, 143/154, 143/213/233, 143/213/233/238/261/343, 143/213/261/343, 143/233/261 , 143/261 , 151/154, 151/154/213/343, 151/154/343, 151/213, 151/213/233, 213/233/343, and 233/238/343 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4984. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5186. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5186, wherein the C-terminal histidine tag is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5186, wherein the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5354. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5354, wherein the C-terminal histidine tag is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5354, wherein the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof is replaced or removed.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 117, 117/118/143/168/169/392, 117/118/143/392, 117/118/168/169, 117/118/168/169/392, 117/143/168/169/170/392, 117/143/168/169/392, 117/168/169, 117/168/169/392, 117/168/392, 117/169, 117/392, 118/143, 118/143/168/169, 118/143/169, 118/143/392, 118/168, 118/168/392, 118/169/392, 118/392, 143/168/169/392, 143/169, 143/392, 168/169, 168/169/392, 168/392, 169/392, and 392 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5196.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 134, 134/182/194/294, 134/182/277/293, 134/182/293/320, 134/182/294, 134/194/277, 134/194/277/317, 134/194/293, 134/194/293/294, 134/194/293/317, 134/194/294, 134/194/317, 134/194/317/320, 134/317, 182, 182/194, 182/194/293/320, 182/293/294, 194, 194/277/293/317, 194/277/317/320, 194/277/320, 194/293/294/317, 194/293/320, 277, 293, 317, 317/320, and 320 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5286.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 12/134, 13/134, 13/134/210, 22/134/152, 22/134/235/344, 64/93/134/235, 64/134/344/347, 77/134, 79/134/210, 84/134, 86/134, 87/134, 89/134, 93/134/152/235/344, 93/134/235/344, 93/134/244/344, 93/134/344, 131 , 134/152/344, 134/175, 134/210/382, 134/210/402, 134/210/456, 134/210/478, 134/344, 134/377, 134/378, 134/380, 134/381 , 134/382, 134/384, 134/386, 134/387, 134/388, 134/390, 134/391 , 134/393, 134/395, 134/398, 134/401 , 134/402, 134/406, 134/407, 134/421 , 134/456, 134/457, 134/459, 134/460, 134/464, 134/467, 134/468, 134/470, 134/471 , 134/473, 134/473/474, 134/474, and 134/475 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5412.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 73/127/134/160/172/198/290, 73/127/134/160/198/333, 73/127/134/160/210/266, 73/127/134/172/192/266/333, 73/127/134/172/198, 73/127/134/210/290, 73/127/134/210/333, 73/127/134/290/333, 73/127/134/333, 73/134, 73/134/160/172/192/290, 73/134/160/172/198/333, 73/134/160/172/290, 73/134/160/266, 73/134/160/333, 73/134/172, 73/134/172/210/333, 73/134/172/266/333, 73/134/198/210, 73/134/198/210/266, 73/134/210/333, 73/134/333, 73/134/366, 127/134, 127/134/160/172/192/333, 127/134/160/172/210/266/290, 127/134/172/198/266/333, 127/134/192/210, 127/134/198/210/290, 127/134/210/266/290, 127/134/210/333, 134, 134/160/172/333, 134/160/198/266/290, 134/172/192/290/333, 134/192/198, 134/198, 134/198/266/333, 134/210, 134/210/290, and 134/333 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5412. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5500. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5500, wherein the C-terminal histidine tag is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5500, wherein the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5520. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5520, wherein the C-terminal histidine tag is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5520, wherein the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof is replaced or removed.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from 13, 13/178/381 , 13/178/381/473, 13/178/457, 13/381 , 13/381/457, 13/402/457, 13/457, 178, 178/381/457, 178/381/457/473, 178/402/457, 178/473, 381 , 381/393/457, 381/457/473, 381/473, and 393/457 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5422.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from the reference sequence is SEQ ID NO: 5704 and the at least one substitution comprises substitutions at amino acid positions selected from 48, 116 ,118, 120, 130, 137, 153, 157, 163, 175, 181 , 187, 192, 213, 246, 263, 273, 296, 318, 334, 341 , 394, 412, 424, 454, and 458 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5704. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5738. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5738, wherein the C-terminal histidine tag is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5738, wherein the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof is replaced or removed.
In some further embodiments, the engineered transposase comprises a polypeptide sequence comprising at least one substitution or substitution set comprising substitutions at amino acid positions selected from
38/73/113/115/117/118/127/134/152/154/160/163/164/168/169/172/184/192/194/198/220/243/260/266/27 0/295/317/333/392/423,
38/73/113/115/117/118/127/134/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/29 5/317/333/392/423,
38/73/113/115/117/118/127/134/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/29 5/317/333/392/423/457,
38/73/113/115/117/118/127/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/295/31 7/333/392/423,
38/73/113/115/118/127/152/154/160/162/164/168/172/184/192/198/220/243/260/266/270/295/317/333/42 3,
38/73/113/115/118/127/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/295/317/33 3/423,
38/73/113/115/118/127/152/154/160/164/168/172/184/189/192/198/220/243/260/266/270/295/317/333/42 3,
38/73/113/115/118/127/152/154/160/164/168/172/184/192/198/210/220/243/260/266/270/295/317/333/42 3, 38/73/113/115/118/127/152/154/160/164/168/172/184/192/198/220/243/260/266/270/295/317/333/423, 38/73/113/118/127/152/154/160/164/168/172/184/192/198/220/243/260/266/270/295/317/333/423, 38/73/113/118/127/152/154/160/164/168/172/184/192/198/220/243/266/270/317/333/423, 38/73/113/118/127/152/154/160/164/168/172/184/192/198/220/266/270/317/333/423, and 38/113/127/152/154/160/164/168/172/198/220/290/317/423, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5702. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5702, wherein the C-terminal histidine tag is replaced or removed. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5702, wherein the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof is replaced or removed.
In some additional embodiments, the present invention provides engineered transposases capable of binding to adapters or donor DNA to form a transposome, cleaving or fragmenting target DNA, and/or ligating the adapters or donor DNA to the target DNA.
In some further embodiments, the present invention provides engineered transposases have at least one improved property, as compared to a wild-type or reference transposase. In some embodiments, the improved property is selected from increased polynucleotide fragmentation or cleavage activity, increased thermostability, increased activity at elevated temperatures, increased soluble expression, decreased inhibition, increased adapter loading or adapter ligation (i.e. tagging), increased binding to a target polynucleotide, and/or decreased sequence insertion bias.
In still some additional embodiments, the present invention provides engineered transposases comprise increased polynucleotide fragmentation or cleavage activity as compared to a wild-type or reference transposase.
In yet some additional embodiments, the engineered transposases comprise increased thermal stability as compared to a wild-type or reference transposase. In some further embodiments, the engineered transposase comprises increased activity at elevated temperatures as compared to a wildtype or reference transposase.
In some further embodiments, the engineered transposases comprise increased adapter loading or adapter ligation (i.e. tagging) as compared to a wild-type or reference transposase.
In some additional embodiments, the engineered transposases comprise increased binding to a target polynucleotide, as compared to a wild-type or reference transposase.
In yet some further embodiments, the engineered transposases comprise decreased sequence insertion bias, as compared to a wild-type or reference transposase.
In some additional embodiments, the engineered polypeptide comprises an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater sequence identity to any polypeptide sequence set forth in SEQ ID NOs: 2-2368, 2387-5694, and 5706-5756.
In some additional embodiments, the engineered transposase comprises a polypeptide sequence selected from the polypeptide sequences between SEQ ID NOs: 2-2368, 2388-5694, and 5706-5756.
In yet some additional embodiments, the present invention provides purified engineered transposases.
The present invention also provides compositions comprising at least one engineered transposase provided herein.
The present invention also provides engineered polynucleotide sequences encoding at least one engineered transposase provided herein.
In some embodiments, the present invention provides engineered polynucleotide sequences comprising at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NOs: 1 , 3, 155, 301 , 337, 605, 971 , 1081 , 1217, 1579, 1597, 1599, 1603, 1719, 2367, 2425, 2539, 3401 , 3661 , 3787, 4149, 4277, 4641 , 4983, 5195, 5285, 5411 , 5421 , and/or 5703-5755, and/or or a functional fragment thereof, wherein said polynucleotide sequence encodes an engineered polypeptide comprising at least one substitution at one or more amino acid positions. In some embodiments, a functional fragment of an engineered polynucleotide sequence does not contain the segment of nucleotides encoding the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) and/or the 6-hisitine tag.
In some further embodiments, the engineered polynucleotide sequences encode at least one engineered transposase comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704. In some additional embodiments, the engineered polynucleotide sequence comprises SEQ ID NOs: 1 , 3, 155, 301 , 337, 605, 971 , 1081 , 1217, 1579, 1597, 1599, 1603, 1719, 2367, 2425, 2539, 3401 , 3661 , 3787, 4149, 4277, 4641 , 4983, 5195, 5285, 5411 , 5421 , and/or 5705-5755.
In yet some additional embodiments, the engineered polynucleotide sequence is operably linked to a control sequence.
In still some other embodiments, the engineered polynucleotide sequence is codon-optimized.
The present invention also provides an engineered polynucleotide encoding at least one engineered polypeptide described in the above paragraphs. In some embodiments, the engineered polynucleotide comprises the polynucleotide sequences set forth in SEQ ID NOs: 1 -2367, SEQ ID NOs: 2385-5693, and SEQ ID NOs: 5705-5755. In some embodiments, the engineered polynucleotide comprises the polynucleotide sequences set forth in SEQ ID NO: 2395. In some embodiments, the engineered polynucleotide comprises the polynucleotide sequences set forth in SEQ ID NO: 4641 . In some embodiments, the engineered polynucleotide comprises the polynucleotide sequences set forth in SEQ ID NO: 4871 . In some embodiments, the engineered polynucleotide comprises the polynucleotide sequences set forth in SEQ ID NO: 4953. In some embodiments, the engineered polynucleotide comprises the polynucleotide sequences set forth in SEQ ID NO: 5185. In some embodiments, the engineered polynucleotide comprises the polynucleotide sequences set forth in SEQ ID NO: 5353. In some embodiments, the engineered polynucleotide comprises the polynucleotide sequences set forth in SEQ ID NO: 5499. In some embodiments, the engineered polynucleotide comprises the polynucleotide sequences set forth in SEQ ID NO: 5519. In some embodiments, the engineered polynucleotide comprises the polynucleotide sequences set forth in SEQ ID NO: 5701 . In some embodiments, the engineered polynucleotide comprises the polynucleotide sequences set forth in SEQ ID NO: 5737. In some embodiments, the engineered polynucleotide comprises one of the foregoing polynucleotide sequence or a polynucleotide sequence encoding an engineered polypeptide described herein (e.g., an engineered transposase), wherein the segment of nucleotides encoding the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) and/or the 6-histidine tag is modified or removed. In some embodiments, the segment of nucleotides encoding the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) is modified or replaced to encode a different C-terminal peptide spacer. In some embodiments, the segment of nucleotides encoding the C-terminal 6-histidine tag is modified or replaced to encode a different tag. In some embodiments, the segment of nucleotides encoding the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) and/or the 6-histidine tag is removed.
The present invention further provides vectors comprising at least one engineered polynucleotide described above. In some embodiments, the vectors further comprise at least one control sequence.
The present invention also provides host cells comprising the vectors provided herein. In some embodiments, the host cell produces at least one engineered polypeptide provided herein.
The present invention further provides methods of producing an engineered transposase polypeptide, comprising the steps of culturing the host cell provided herein under conditions such that the engineered polynucleotide is expressed and the engineered polypeptide is produced. In some embodiments, the methods further comprise the step of recovering the engineered polypeptide. In some embodiments, the methods further comprise the step of purifying at least one engineered transposase. In some embodiments, the invention provides, a method for generating a mixture of altered nucleic acids comprising exposing a target nucleic acid to an engineered transposase under conditions and for a time sufficient for the engineered transposase to carry out a transposition event, wherein the engineered transposase comprises a polypeptide sequence comprising at least 85% sequence identity to a reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, or a functional fragment thereof, wherein the engineered transposase comprises at least one substitution or substitution set in its polypeptide sequence relative to the reference sequence.
In some embodiments, the altered nucleic acids are fragmented from the target nucleic acid.
In some embodiments, the invention provides, a method of preparing a nucleic acid library comprising:
(a) contacting a target nucleic acid with an engineered transposase under conditions and for a time sufficient for the engineered transposase to carry out a transposition event, wherein the engineered transposase comprises a polypeptide sequence comprising at least 85% sequence identity to a reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, or a functional fragment thereof, wherein the engineered transposase comprises at least one substitution or substitution set in its polypeptide sequence relative to the reference sequences;
(b) fragmenting the target nucleic acid into a plurality of nucleic acid fragments;
(c) selecting a subset of nucleic acid fragments from the plurality of nucleic acid fragments; and
(d) amplifying the subset of nucleic acid fragments to generate the nucleic acid library.
In some embodiments, the invention provides, a method of sequencing a target nucleic acid comprising:
(a) contacting the target nucleic acid with an engineered transposase under conditions and for a time sufficient for the engineered transposase to carry out a transposition event, wherein the engineered transposase comprises a polypeptide sequence comprising at least 85% sequence identity to a reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, or a functional fragment thereof, wherein the engineered transposase comprises at least one substitution or substitution set in its polypeptide sequence relative to the reference sequence;
(b) fragmenting the target nucleic acid into a plurality of nucleic acid fragments;
(c) selecting a subset of nucleic acid fragments from the plurality of nucleic acid fragments;
(d) amplifying the subset of nucleic acid fragments to generate a nucleic acid library; and
(e) sequencing the nucleic acid library.
In some embodiments, the method further includes adding a polynucleotide to the 5’ and/or 3’ end of each nucleic acid fragment of the plurality of nucleic acid fragments.
In some embodiments, the engineered transposase is any one of the engineered transposases of the foregoing embodiments. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 2396. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4642. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4872. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 4954. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5186. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5354. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5500. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5520. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5702. In some embodiments, the engineered transposase comprises the polypeptide sequence of SEQ ID NO: 5738. In some embodiments, the polypeptide sequence of the engineered transposase is modified to replace or remove the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) and/or the 6-histidine tag. In some embodiments, the polypeptide sequence of the engineered transposase is modified to replace the C-terminal peptide spacer GGSG (SEQ ID NO: 5777) with a different peptide spacer. In some embodiments, the polypeptide sequence of the engineered transposase is modified to replace the 6-histidine tag with a different tag. In some embodiments, the polypeptide sequence of the engineered transposase is modified to remove the C-terminal histidine tag and the peptide spacer GGSG (SEQ ID NO: 5777) or a portion thereof.
In some embodiments, the engineered transposase is purified.
In some embodiments, fragmenting the target nucleic acid comprises tagmentation or random sheering and adapter ligation. In some embodiments, fragmenting the target nucleic acid comprises tagmentation.
In some embodiments, each altered nucleic acid of the mixture of altered nucleic acids comprises an identifiable sequence tag (1ST).
In some embodiments, each nucleic acid fragment of the subset of nucleic acid fragments comprises an identifiable sequence tag (1ST). In some embodiments, the 1ST is between 6 and 30 nucleotides in length (e.g., 6-10, 10-15, 15-20, 20-25, 25-30, such as 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).
In some embodiments, each altered nucleic acid of the mixture of altered nucleic acids is between 30 and 30000 nucleotides in length (e.g., 30-30000, 30-25000, 30-20000, 30-15000, 30-10000, 30-7500, 30-5000, 30-4000, 30-3000, 30-2500, 30-2000, 30-1500, 30-1000, 30-900, 30-800, 30-700, 30-600, 30- 500, 30-400, 30-300, 30-250, 30-200, 30-150, 30-100, 30-50, 100-500, 100-1000, 100-1500, 100-2500, 100-5000, 100-10000, 100-20000, 100-30000, 500-1000, 500-2500, 500-5000, 500-10000, 500-20000, 500-30000, 1000-2500, 1000-5000, 1000-10000, 1000-20000, 1000-30000, 5000-10000, 5000-20000, 5000-30000, 10000-20000, 20000-30000, 2500-5000, 7500-10000, or 15000-25000 nucleotides in length).
In some embodiments, each nucleic acid fragment of the plurality of nucleic acid fragments is between 30 and 30000 nucleotides in length (e.g., 30-30000, 30-25000, 30-20000, 30-15000, 30-10000, 30-7500, 30-5000, 30-4000, 30-3000, 30-2500, 30-2000, 30-1500, 30-1000, 30-900, 30-800, 30-700, 30- 600, 30-500, 30-400, 30-300, 30-250, 30-200, 30-150, 30-100, 30-50, 100-500, 100-1000, 100-1500, 100- 2500, 100-5000, 100-10000, 100-20000, 100-30000, 500-1000, 500-2500, 500-5000, 500-10000, 500- 20000, 500-30000, 1000-2500, 1000-5000, 1000-10000, 1000-20000, 1000-30000, 5000-10000, 5000- 20000, 5000-30000, 10000-20000, 20000-30000, 2500-5000, 7500-10000, or 15000-25000 nucleotides in length).
In some embodiments, the transposition event occurs in reaction conditions additionally comprising a terminal deoxynucleotide transferase, dNTP, and/or buffer components suitable for the addition of deoxynucleotides to the 3’ terminus of each of the plurality of nucleic acid fragments.
In some embodiments, the transposition event occurs in reaction conditions additionally comprising a DNA ligase and/or buffer components suitable for a ligation reaction.
In some embodiments, each altered nucleic acid of the mixture of altered nucleic acids further comprises a sample tag and/or a unique molecular identifier (UMI). In some embodiments, each nucleic acid fragment of the subset of nucleic acid fragments further comprises a sample tag and/or a unique molecular identifier (UMI).
In some embodiments, amplification of the subset of nucleic acid fragments is performed through polymerase chain reaction (PCR), multiple displacement amplification (MDA), ligase chain reaction (LCR), loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), or strand displacement amplification (SDA).
In some embodiments, the sequencing comprises next-generation sequencing (NGS).
In some embodiments, the sequencing comprises sequencing by synthesis, sequencing by ligation, or nanopore sequencing. In some embodiments, the sequencing by synthesis comprises Illumina™ dye sequencing, single-molecule real-time (SMRT™) sequencing, or pyrosequencing. In some embodiments, the sequencing by ligation comprises polony-based sequencing or SOLiD™ sequencing.
In some embodiments, the target nucleic acid comprises genomic DNA or cDNAs from a single cell. In some embodiments, the target nucleic acid comprises nucleic acids from a plurality of haplotypes.
In some embodiments, the target nucleic acid is crosslinked via histones or chromatin from single or multiple cells. In some embodiments, the target nucleic acid has been condensed or optionally treated with one or more condensing agents.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts active transposase variants, bound to appropriate adapter sequences, that are capable of binding target sequences, cleaving the target strand, and ligating the adapter oligonucleotides to the target fragment.
Figure 2 provides a scheme depicting the capillary electrophoresis (CE) assay used in the Examples to assess the activity of the engineered transposase variants.
Figure 3 is a plot of fragment sizes of NGS libraries prepared from 50 ng input DNA sample.
Libraries prepared with a transposase that has the polypeptide sequence set forth in SEQ ID NO: 5704 or a commercially available Tn5 transposase are indicated.
Figures 4A through 4D are plots showing the insertion bias of the indicated transposases as determined by the calculated root mean square error (RMSE) of the first 25 bases of the insert. Figures 5A and 5B are gels showing the results of the enzyme activity assay comparing engineered transposase (SequenceK; Figure 5A) with a commercial transposase (hyperactive Tn5) for their ability to convert circular plasmid to linear form. 2.5 pmol transposase was used in the comparison. Plasmids are present in the gel as the higher length bars, i.e. >3000 bp, while the linear form fragments are shown as the smear < 3000 bp.
Figure 6 is a plot of fragment sizes of NGS libraries prepared from 50 ng - 200 ng input DNA sample.
Figure 7 is a plot showing the estimated NGS library size generated from 100 ng of human NA12878 genomic DNA using different engineered transposases (SequenceE, Sequencer, SequenceG, SequenceH, SequenceK, and SequenceL) and commercial hyperactive Tn5 transposases (Hyp-Tn5).
Figure 8 is a plot showing GC coverage bias of NGS libraries generated from 100 ng of human NA12878 genomic DNA using different engineered transposases (SequenceE; SequenceK; SequenceF; SequenceG; SequenceH; and SequenceL) and commercial hyperactive Tn5 transposases.
Figure 9 is a plot showing fold-coverage of NGS libraries generated from 100 ng of human NA12878 genomic DNA using different engineered transposases (SequenceE; SequenceK; SequenceF; SequenceG; SequenceH; and SequenceL) and commercial hyperactive Tn5 transposases.
Figures 10A through 10D are plots showing the per base sequence content of the transposition sites of the different commercial transposases and engineered transposases. Each nucleotide (A, T, G, or C) is represented by a single line trace, representing the percentage of reads having that nucleotide at the corresponding position relative to the read start site (transposition site) of the DNA molecules in the sequenced library.
Figure 11 are plots showing GC coverage bias of NGS libraries prepared by ExpressPlex using hyperactive Tn5 transposase and the SequenceH engineered transposase from Staphylococcus epidermidis, Bacillus subtilis, Escherichia coll, and Pseudomonas aeruginosa.
Figure 12 is a plot showing the coverage profiles of NGS libraries prepared by ExpressPlex of lambda amplicon 18 DNA samples using a commercial and engineered (SequenceE, SequenceH, and SequenceK) transposases. Data is shown as the raw coverage profile as well as the summary statistic in the form of the coefficient of variation (CV) of the raw coverage profile.
Figure 13 is a plot showing the coverage profiles of NGS libraries prepared by ExpressPlex of pUC19 DNA samples using commercial and engineered (SequenceE, SequenceH, and SequenceK) transposases. Data is shown as the raw coverage profile as well as the summary statistic in the form of the coefficient of variation (CV) of the raw coverage profile.
DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well-known and commonly employed in the art. Such techniques are well-known and described in numerous texts and reference works well known to those of skill in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses. All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.
Although any suitable methods and materials similar or equivalent to those described herein find use in the practice of the present invention, some methods and materials are described herein. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art. Accordingly, the terms defined immediately below are more fully described by reference to the invention as a whole.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present invention. The section headings used herein are for organizational purposes only and not to be construed as limiting the subject matter described. Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.
As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a polypeptide” includes more than one polypeptide. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.
It is to be understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.” It is to be further understood that where descriptions of various embodiments use the term “optional” or “optionally” the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. It is to be understood that both the foregoing general description, and the following detailed description are exemplary and explanatory only and are not restrictive of this disclosure. The section headings used herein are for organizational purposes only and not to be construed as limiting the subject matter described.
Abbreviations:
The abbreviations used for the genetically encoded amino acids are conventional and are as follows:
When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D- configuration about a-carbon (Ca). For example, whereas “Ala” designates alanine without specifying the configuration about the a-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively.
When the one-letter abbreviations are used, upper case letters designate amino acids in the L- configuration about the a-carbon and lower case letters designate amino acids in the D-configuration about the a-carbon. For example, “A” designates L-alanine and “a” designates D-alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.
The abbreviations used for the genetically encoding nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). These abbreviations are also used interchangeably for nucleosides and nucleotides (nucleosides with one or more phosphate groups). Unless specifically delineated, the abbreviated nucleosides or nucleotides may be either ribonucleosides (or ribonucleotides) or 2’-deoxyribonucleosides (or 2’-deoxyribonucleotides). The nucleosides or nucleotides may also be modified at the 3’ position. The nucleosides or nucleotides may be specified as being either ribonucleosides (or ribonucleotides) or 2’-deoxyribonucleosides (or 2’- deoxyribonucleotides) on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5’ to 3’ direction in accordance with common convention, and the phosphates are not indicated.
Definitions:
In reference to the present invention, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings.
“EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.
“ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.
“NCBI” refers to National Center for Biological Information and the sequence databases provided therein.
“Protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids, as well as polymers comprising D- and L-amino acids, and mixtures of D- and L-amino acids.
“Amino acids” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.
As used herein, “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably herein and refer to two or more nucleosides or nucleotides that are covalently linked together. The polynucleotide may be wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of 2’ deoxyribonucleotides (i.e., DNA), wholly comprised of other synthetic nucleotides or comprised of mixtures of synthetic, ribo- and/or 2’ deoxyribonucleotides. The polynucleotides may also include modified nucleotides with substitutions, including 2’ substitutions (e.g., 2’-fluoro, 2’-O-methyl, 2’-O-methoxyethyl, locked or constrained ethyl modifications, and others known to those skilled in the art). Nucleosides will be linked together via standard phosphodiester linkages or via one or more non-standard linkages, including but not limited to phosphorothioate linkages. The polynucleotide may be single-stranded or doublestranded or may include both single-stranded regions and double-stranded regions. Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine and cytosine), it may include one or more modified and/or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino-acid sequences. Nucleobases that are modified or synthetic may comprise any known or hypothetical or future discovered modification or structure that would be recognized by one of skill in the art as a modified or synthetic nucleobase. Examples of modified nucleobases or nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouraci 1 , 5-methyluracil, uracil-5-oxyacetic acid methylester, 5-methyl- 2-thiouracil, and 3-(3-amino-3-N-2-carboxypropyl) uracil. A polynucleotide may contain one or more chemical modifications, including modifications on the 5’ end, the 3’ end, or internally. Examples of chemical modifications include, but are not limited to, addition of functional groups (e.g., biotins, amino modifiers, alkynes, thiol modifiers, or azides), fluorophores (e.g. quantum dots or organic dyes), spacers (e.g. C3 spacer, dSpacer, photo-cleavable spacers), modified bases, or modified backbones.
Similarly, the terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are intended to comprise any modified or synthetic structure that is now known or discovered in the future that would be recognized by one of skill in the art as being or having the function of a “polynucleotide,” “oligonucleotide,” or “nucleic acid.” An example of a modified or synthetic structure having the function of a “polynucleotide,” “oligonucleotide,” or “nucleic acid” is PNA or peptide nucleic acid.
“Coding sequence” refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.
“Naturally-occurring” or “wild-type” refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.
As used herein, “recombinant,” “engineered,” and “non-naturally occurring” when used with reference to a cell, nucleic acid, or polypeptide, refer to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature. In some embodiments, the cell, nucleic acid or polypeptide is identical to a naturally occurring cell, nucleic acid or polypeptide, but is produced or derived from synthetic materials and/or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or expressed native genes that are otherwise expressed at a different level.
“Percentage of sequence identity” and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides or polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482 [1981]), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443 [1970]), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (See, Altschul et al., J. Mol. Biol., 215: 403-410 [1990]; and Altschul et al., Nucl. Acids Res., 3389-3402 [1977], respectively). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11 , an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1989]). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wl), using default parameters provided.
“Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1 ) comprise a sequence {i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence. For instance, a “reference sequence based on SEQ ID NO:4 having at the residue corresponding to X14 a valine” or X14V refers to a reference sequence in which the corresponding residue at X14 in SEQ ID NO:4, which is a tyrosine, has been changed to valine.
“Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions {i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.
As used herein, “substantial identity” refers to a polynucleotide or polypeptide sequence that has at least 80 percent sequence identity, at least 85 percent identity, at least between 89 to 95 percent sequence identity, or more usually, at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 residue positions, frequently over a window of at least 30-50 residues, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions which total 20% or less of the reference sequence over the window of comparison. In some specific embodiments applied to polypeptides, the term “substantial identity” means that two polypeptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80% sequence identity, preferably at least 89% sequence identity, at least 95% sequence identity or more (e.g., 99% sequence identity). In some embodiments, residue positions that are not identical in sequences being compared differ by conservative amino acid substitutions.
“Corresponding to,” “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered transposase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned. “Amino acid difference” or “residue difference” refers to a change in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, a “residue difference at position X25 as compared to SEQ ID NO: 2” refers to a change of the amino acid residue at the polypeptide position corresponding to position 25 of SEQ ID NO:2. Thus, if the reference polypeptide of SEQ ID NO: 2 has a valine at position 25, then a “residue difference at position X25 as compared to SEQ ID NO:2” an amino acid substitution of any residue other than valine at the position of the polypeptide corresponding to position 25 of SEQ ID NO: 2. In most instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding position as described above, and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some embodiments, more than one amino acid can appear in a specified residue position (i.e., the alternative amino acids can be listed in the form XnY/Z, where Y and Z represent alternate amino acid residues). In some instances (e.g., in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 10.3, 10.5, 11 .2, 11 .4, 11 .5, 12.2, 12.3, 12.5, 12.6, 13.2, 13.3, 13.4, 14.2, 14. 3, 14.5, 14.6, 14.8, 14.9, 15.1 , 16.2, 16.3, 17.2, 17.3, 18.2,
19.2, 19.3, 20.2, 20.3, 21 .2, 21 .3, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31 .2, 32.2, 33.2,
34.2, 35.1 , 36.1 , and 36.3) the present invention also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. Furthermore, in some instances, a polypeptide of the present invention can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where changes are made relative to the reference sequence. In some additional embodiments, the present invention provides engineered polypeptide sequences comprising both conservative and non-conservative amino acid substitutions.
As used herein, “conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with an hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively. Exemplary conservative substitutions are provided in Table 1.
“Non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.
“Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and/or retaining the improved properties of an engineered transposase enzyme. Deletions can be directed to the internal portions and/or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous.
“Insertion” refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. In some embodiments, the improved engineered transposase enzymes comprise insertions of one or more amino acids to the naturally occurring polypeptide as well as insertions of one or more amino acids to other improved transposase polypeptides. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.
“Fragment” as used herein refers to a polypeptide that has an amino-terminal and/or carboxyterminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence. Fragments can be at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long or longer, and up to 70%, 80%, 90%, 95%, 98%, and 99% of the full-length transposase polypeptide, for example the polypeptide of SEQ ID NO: 2 or a transposase provided in the polypeptide sequences of SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756. “Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system {e.g., host cell or in vitro synthesis). The engineered transposase enzymes may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the engineered transposase enzyme can be an isolated polypeptide.
“Substantially pure polypeptide” refers to a composition in which the polypeptide species is the predominant species present (/.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition) and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure transposase composition will comprise about 60 % or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (/.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated engineered transposase polypeptide is a substantially pure polypeptide composition.
As used herein, a “target nucleic acid” refers to any nucleic acid (e.g., RNA or DNA) of interest that is selected for amplification or analysis (e.g., sequencing) using a composition (e.g., sequencing oligonucleotides or barcoding oligonucleotides) or method of the invention. In some instances, RNA may be converted to cDNA prior to being used in the methods described herein. In some instances, target nucleic acid includes genomic DNA or cDNAs from a single cell. In some instances, target nucleic acid includes nucleic acids from a plurality of haplotypes. In some instances, target nucleic acid is crosslinked via histones or chromatin from single or multiple cells. In some instances, target nucleic acid has been condensed or optionally treated with one or more condensing agents.
As used herein, “improved enzyme property” refers to at least one improved property of an enzyme. In some embodiments, the present invention provides engineered transposase polypeptides that exhibit an improvement in any enzyme property as compared to a reference transposase polypeptide and/or a wild-type transposase polypeptide, and/or another engineered transposase polypeptide. Thus, the level of “improvement” can be determined and compared between various transposase polypeptides, including wild-type, as well as engineered transposases. Improved properties include, but are not limited, to such properties as increased polynucleotide fragmentation or cleavage activity, increased thermostability, increased activity at elevated temperatures, increased soluble expression, decreased inhibition, increased adapter loading (donor binding) or adapter ligation (i.e. tagging), increased binding to a target polynucleotide, and/or decreased sequence insertion bias as compared to another wild-type transposase or engineered transposase.
“Sequence insertion bias,” “target bias,” “sequence bias,” or related terms used herein refer to the degree to which a transposome cleaves target nucleotide sequences in a non-random manner based on the composition of the sequence of the polynucleotide. For example, a transposase with G/C target bias will have an increased rate of binding to G/C rich sequences, as compared to other sequences. As detailed in the Examples below, sequence insertion bias can be approximated or quantified using several methods. One method evaluates bias over the first 15 nucleotides of NGS reads putatively comprising the region of the target bound by the transposome construct. Bias plots generated from NGS data represent this bias as a function of the distance from the insertion site. Overall relative bias for each variant can be calculated as the root-mean-squared deviation (RMSD) of the observed vs. expected representation of all four nucleotides at each position. Conversely, “insertion promiscuity” and “promiscuity” refer to the degree to which a transposome binds to target nucleotide sequences in a random manner, without regard to the sequence composition of the polynucleotide.
“Next generation sequencing” refers to modern high-throughput or massively-parallel sequencing techniques that allow sequencing of many polynucleotide sequences at the same time, as compared to traditional sequencing methods, including the Sanger method, that allowed sequencing of only one polynucleotide at a time. Next generation sequencing methods have facilitated whole genome sequencing and enabled genomics and related fields by requiring less polynucleotide sample and having greater accuracy.
“Adapter,” as used herein, refers to a donor polynucleotide that contains the end sequences for transposase binding, as well as other optional application-specific elements. These may include binding sites for sequencing primers, library amplification primers, sample indexes, or other functional donor polynucleotide sequence. The adapter is loaded onto or bound to the transposase, which dimerizes to form the “transposome”.
“Barcode,” as used herein, refers to a unique oligonucleotide sequence that may allow the corresponding oligonucleotide to be identified. In some instances, the nucleic acid sequence may be located at a specific position in a longer nucleic acid sequence.
“Complement” or “complementary,” as used herein in reference to a sequence, refers to the sequence of a first nucleic acid in relation to that of a second nucleic acid, wherein when the first and second nucleic acids are aligned antiparallel (5’ end of the first nucleic acid matched to the 3’ end of the second nucleic acid, and vice versa) to each other, the nucleotide bases at each position in their sequences will have complementary structures following a lock-and-key principle (i.e. , A will be paired with U or T and G will be paired with C). Complementary sequences may include mismatches of up to one third of nucleotide bases. For example, two sequences that are nine bases in length may have mismatches of at most 3, at most 2, or at most 1 , or at most 0 nucleotide bases, and remain complementary to one another.
“Transposition” or a “transposition event,” as used herein, is the cleavage of a target dsDNA (or hybrid DNA/RNA) sequence and ligation of the pre-loaded adapter (or another sequence containing transposon end sequences) from the transposome onto the target dsDNA at the site of cleavage. In cells, transposition typically refers to the relocation of a segment of DNA to another locus in the genome. In vitro, transposition may result in the fragmentation of a target DNA sample with the simultaneous addition of the adapter sequences to the fragmented DNA ends, i.e., tagmentation. “Synaptic complex” or “transposase synaptic complex,” as used herein, refers to a protein-nucleic acid complex including one or more transposases and one or more oligonucleotides. In some instances, the one or more oligonucleotides of the synaptic complex are inserted into a nucleic acid sequence of a nucleic acid sample by transposase activity. In some instances, the synaptic complex may include two transposases and two oligonucleotides. In some instances, the insertion of the two oligonucleotides into the nucleic acid sequence of the nucleic acid sample results in fragmentation of the nucleic acid sequence at the site of insertion. In some instances, the transposases may be Tn5 transposases. In some instances, the oligonucleotides may be adapter sequences. In some instances, the synaptic complex is pre-assembled.
“Increased enzymatic activity” refers to an improved property of the transposase polypeptides, which can be represented by an increase in specific activity {e.g., product produced/time/weight protein) or an increase in percent conversion of the substrate to the product {e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of transposase) as compared to the reference transposase enzyme. Exemplary methods to determine enzyme activity are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of Km, Vmax or kcat, changes of which can lead to increased enzymatic activity. Improvements in enzyme activity can be from about 1 .2 times the enzymatic activity of the corresponding wild-type enzyme, to as much as 2 times, 5 times, 10 times, 20 times, 25 times, 50 times or more enzymatic activity than the naturally occurring or another engineered transposase from which the transposase polypeptides were derived. Transposase activity can be measured by any one of standard assays, such as by monitoring changes in properties of substrates, cofactors, or products. In some embodiments, the amount of products generated can be measured by Liquid Chromatography-Mass Spectrometry (LC-MS), HPLC, capillary electrophoresis or other methods, as known in the art. Comparisons of enzyme activities are made using a defined preparation of enzyme, a defined assay under a set condition, and one or more defined substrates, as further described in detail herein. Generally, when lysates are compared, the numbers of cells and the amount of protein assayed are determined as well as use of identical expression systems and identical host cells to minimize variations in amount of enzyme produced by the host cells and present in the lysates.
“Conversion” refers to the enzymatic conversion of the substrate(s) to the corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzymatic activity” or “activity” of a transposase polypeptide can be expressed as “percent conversion” of the substrate to the product.
“Thermostable” refers to a polypeptide that maintains similar activity (more than 60% to 80% for example) after exposure to elevated temperatures {e.g., 40-80 °C) for a period of time {e.g., 0.5-24 hrs) compared to the wild-type enzyme exposed to the same elevated temperature.
“Solvent stable” refers to a polypeptide that maintains similar activity (more than e.g., 60% to 80%) after exposure to varying concentrations {e.g., 5-99%) of solvent (ethanol, isopropyl alcohol, dimethylsulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time {e.g., 0.5-24 hrs) compared to the wild-type enzyme exposed to the same concentration of the same solvent. “Thermo- and solvent stable” refers to a polypeptide that is both thermostable and solvent stable. The term “stringent hybridization conditions” is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrids. In general, the stability of a hybrid is a function of ion strength, temperature, G/C content, and the presence of chaotropic agents. The Tm values for polynucleotides can be calculated using known methods for predicting melting temperatures (See e.g., Baldino et al., Meth. Enzymol., 168:761 -777 [1989]; Bolton et al., Proc. Natl. Acad. Sci. USA 48:1390 [1962]; Bresslauer et al., Proc. Natl. Acad. Sci. USA 83:8893-8897 [1986]; Freier et al., Proc. Natl. Acad. Sci. USA 83:9373-9377 [1986]; Kierzek et al., Biochem., 25:7840-7846 [1986]; Rychlik et al., 1990, Nucl. Acids Res., 18:6409-6412 [1990] (erratum, Nucl. Acids Res., 19:698 [1991 ]); Sambrook et al., supra); Suggs et al., 1981 , in Developmental Biology Using Purified Genes, Brown et al. [eds.], pp. 683-693, Academic Press, Cambridge, MA [1981]; and Wetmur, Crit. Rev. Biochem. Mol. Biol., 26:227-259 [1991]). In some embodiments, the polynucleotide encodes the polypeptide disclosed herein and hybridizes under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered transposase enzyme of the present invention.
“Hybridization stringency” relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency. The term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in 0.2xSSPE, 0.2% SDS, at 42 °C. “High stringency hybridization” refers generally to conditions that are about 10 °C or less from the thermal melting temperature Tm as determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCI at 65 °C (/.e., if a hybrid is not stable in 0.018M NaCI at 65 °C, it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in 0.1 xSSPE, and 0.1 % SDS at 65 °C. Another high stringency condition is hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0.1% (w:v) SDS at 65 °C and washing in 0.1 x SSC containing 0.1 % SDS at 65 °C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.
“Heterologous” polynucleotide refers to any polynucleotide that is introduced into a host cell by laboratory techniques, and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.
“Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the transposase enzymes may be codon optimized for optimal production from the host organism selected for expression.
As used herein, “preferred, optimal, high codon usage bias codons” refers interchangeably to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (See e.g., GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, Peden, University of Nottingham; McInerney, Bioinform., 14:372-73 [1998]; Stenico et al., Nucl. Acids Res., 222437-46 [1994]; Wright, Gene 87:23-29 [1990]). Codon usage tables are available for many different organisms (See e.g., Wada et al., Nucl. Acids Res., 20:2111 -2118 [1992]; Nakamura et al., Nucl. Acids Res., 28:292 [2000]; Duret, et al., supra; Henaut and Danchin, in Escherichia co// and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington D.C., p. 2047-2066 [1996]). The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (See e.g., Mount, Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [ 2001]; Uberbacher, Meth. Enzymol., 266:259-281 [1996]; and Tiwari et al., Comput. Appl. Biosci., 13:263- 270 [1997]).
“Control sequence” is defined herein to include all components, which are necessary or advantageous for the expression of a polynucleotide and/or polypeptide of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.
“Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed (/.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and/or polypeptide of interest. “Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
“Suitable reaction conditions” refer to those conditions in the biocatalytic reaction solution {e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffers, co-solvents, etc.) under which a transposase polypeptide of the present invention is capable of converting one or more substrate compounds to a product compound {e.g., ligation or annealing of an adapter or donor polynucleotide and a target polynucleotide to form an annealed or ligated polynucleotide consisting of the adapter and the optionally fragmented target polynucleotide). Exemplary “suitable reaction conditions” are provided in the present invention and illustrated by the Examples.
“Composition” refers to a mixture or combination of one or more substances, wherein each substance or component of the composition retains its individual properties. As used herein, a biocatalytic composition refers to a combination of one or more substances useful for biocatalysis.
“Loading”, such as in “compound loading” or “enzyme loading” or “cofactor loading” refers to the concentration or amount of a component in a reaction mixture at the start of the reaction. “Adapter loading” refers to binding of the transposase to the adapter polynucleotide.
“Substrate” in the context of a biocatalyst mediated process refers to the compound or molecule acted on by the biocatalyst. For example, a transposase biocatalyst used in the synthesis processes disclosed herein acts on an adapter or donor polynucleotide and a target polynucleotide.
“Product” in the context of a biocatalyst mediated process refers to the compound or molecule resulting from the action of the biocatalyst. For example, an exemplary product for a transposase biocatalyst used in a process disclosed herein is an annealed or ligated polynucleotide consisting of the adapter and the optionally fragmented target polynucleotide, as depicted in Figures 1 and 2.
“Alkyl” refers to saturated hydrocarbon groups of from 1 to 18 carbon atoms inclusively, either straight chained or branched, more preferably from 1 to 8 carbon atoms inclusively, and most preferably 1 to 6 carbon atoms inclusively. An alkyl with a specified number of carbon atoms is denoted in parenthesis (e.g., (Ci-Ce)alkyl refers to an alkyl of 1 to 6 carbon atoms).
“Alkenyl” refers to hydrocarbon groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one double bond but optionally containing more than one double bond.
“Alkynyl” refers to hydrocarbon groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one triple bond but optionally containing more than one triple bond, and additionally optionally containing one or more double bonded moieties.
“Heteroalkyl, “heteroalkenyl,” and heteroalkynyl,” refer respectively, to alkyl, alkenyl and alkynyl as defined herein in which one or more of the carbon atoms are each independently replaced with the same or different heteroatoms or heteroatomic groups. Heteroatoms and/or heteroatomic groups which can replace the carbon atoms include, but are not limited to -O-, -S-, -S-O-, -NR9-, -PH-, -S(O)-, -S(O)2-, - S(0) NR9-, -S(O)2NR9, and the like, including combinations thereof, where each R9 is independently selected from hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.
“Amino” refers to the group -NH2. Substituted amino refers to the group -NHRh, NRhRh, and NRhRhRh, where each Rh is independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Typical amino groups include, but are limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino, furanyl-oxy-sulfamino, and the like.
“Aminoalkyl” refers to an alkyl group in which one or more of the hydrogen atoms are replaced with one or more amino groups, including substituted amino groups.
“Aminocarbonyl” refers to -C(O)NH2. Substituted aminocarbonyl refers to -C(O)NRhRh, where the amino group NRhRh is as defined herein.
“Oxy” refers to a divalent group -O-, which may have various substituents to form different oxy groups, including ethers and esters.
“Alkoxy” or “alkyloxy” are used interchangeably herein to refer to the group -ORZ, wherein Rz is an alkyl group, including optionally substituted alkyl groups.
“Carboxy” refers to -COOH.
“Carbonyl” refers to -C(O)-, which may have a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones.
“Carboxyalkyl” refers to an alkyl in which one or more of the hydrogen atoms are replaced with one or more carboxy groups.
“Aminocarbonylalkyl” refers to an alkyl substituted with an aminocarbonyl group, as defined herein.
“Halogen” or “halo” refers to fluoro, chloro, bromo and iodo.
“Haloalkyl” refers to an alkyl group in which one or more of the hydrogen atoms are replaced with a halogen. Thus, the term “haloalkyl” is meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls. For example, the expression “(Ci - C2) haloalkyl” includes 1 -fluoromethyl, difluoromethyl, trifluoromethyl, 1 -fluoroethyl, 1 ,1 -difluoroethyl, 1 ,2-difluoroethyl, 1 ,1 ,1 trifluoroethyl, perfluoroethyl, etc.
“Hydroxy” refers to -OH.
“Hydroxyalkyl” refers to an alkyl group in which in which one or more of the hydrogen atoms are replaced with one or more hydroxy groups.
“Thiol” or “sulfanyl” refers to -SH. Substituted thiol or sulfanyl refers to -S-Rh, where Rh is an alkyl, aryl or other suitable substituent.
“Sulfonyl” refers to -SO2-. Substituted sulfonyl refers to -SO2-Rh, where Rh is an alkyl, aryl or other suitable substituent.
“Alkylsulfonyl" refers to -SO2-RZ, where Rz is an alkyl, which can be optionally substituted. Typical alkylsulfonyl groups include, but are not limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and the like. “Phosphate” as used herein refers to a functional group comprised of an orthophosphate ion (phosphorous atom covalently linked to four oxygen atoms). The orthophosphate ion is commonly found with one or more hydrogen atoms or organic groups.
“Phosphorylated” as used herein refers to the addition or presence of one of more phosphoryl groups (phosphorous atom covalently linked to the three oxygen atoms).
“Optionally substituted” as used herein with respect to the foregoing chemical groups means that positions of the chemical group occupied by hydrogen can be substituted with another atom (unless otherwise specified) exemplified by, but not limited to carbon, oxygen, nitrogen, or sulfur, or a chemical group, exemplified by, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido, cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; where preferred heteroatoms are oxygen, nitrogen, and sulfur. Additionally, where open valences exist on these substitute chemical groups they can be further substituted with alkyl, cycloalkyl, aryl, heteroaryl, and/or heterocycle groups, that where these open valences exist on carbon they can be further substituted by halogen and by oxygen-, nitrogen-, or sulfur-bonded substituents, and where multiple such open valences exist, these groups can be joined to form a ring, either by direct formation of a bond or by formation of bonds to a new heteroatom, preferably oxygen, nitrogen, or sulfur. It is further contemplated that the above substitutions can be made provided that replacing the hydrogen with the substituent does not introduce unacceptable instability to the molecules of the present invention, and is otherwise chemically reasonable. One of ordinary skill in the art would understand that with respect to any chemical group described as optionally substituted, only sterically practical and/or synthetically feasible chemical groups are meant to be included. “Optionally substituted” as used herein refers to all subsequent modifiers in a term or series of chemical groups. For example, in the term "optionally substituted arylalkyl,” the “alkyl” portion and the “aryl” portion of the molecule may or may not be substituted, and for the series “optionally substituted alkyl, cycloalkyl, aryl and heteroaryl,” the alkyl, cycloalkyl, aryl, and heteroaryl groups, independently of the others, may or may not be substituted.
“Reaction” as used herein refers to a process in which one or more substances or compounds or substrates is converted into one or more different substances, compounds, or processes.
Preparation of Next Generation Sequencing Libraries Using Engineered Transposases
Transposases with improved activity in cleaving or fragmenting polynucleotides and ligating or tagging polynucleotides are necessary to overcome the limitations of existing NGS library preparation methods. Improved transposases with increased polynucleotide fragmentation or cleavage activity, increased thermostability, increased activity at elevated temperatures, increased soluble expression, decreased inhibition, increased adapter loading or adapter ligation (i.e. tagging), increased binding to a target polynucleotide, and/or decreased sequence insertion bias compared to another wild-type or engineered transposase find utility in library preparation for NGS, but are not limited to these uses. The improved transposases of the present disclosure find use in any application involving cleaving or fragmenting double-stranded polynucleotides and/or ligating adapters or tagging polynucleotides, including applications where a donor polynucleotide is transferred into a target polynucleotide.
The present invention provides novel transposases that have improved activity in the fragmentation or cleavage of double stranded DNA and the ligation of adapters or tagging of the DNA fragments. The transposases of the present disclosure have increased polynucleotide fragmentation or cleavage activity, increased thermostability, increased activity at elevated temperatures, increased soluble expression, decreased inhibition, increased adapter loading or adapter ligation (i.e. tagging), increased binding to a target polynucleotide, and/or decreased sequence insertion bias as compared to another wild-type transposase or engineered transposase. The engineered polypeptides of the present disclosure are variants of SEQ ID NO: 2, a chimeric protein based an IS4 transposase from Parashewanella curva or SEQ ID NO: 5704, a modified hyperactive Tn5 transposase, and designed to recognize the19 bp Tn5 mosaic end (ME) transposon donor recognition sequence. These engineered transposases are capable of improved preparation of NGS libraries from polynucleotide samples.
In nature, transposases facilitate the transposition of fragments of DNA (transposons) from a donor strand to a host strand at sites flanked by short inverted-repeat sequences or end sequences (ESs). The transposition mechanism is initiated by the binding of two transposases to donor DNA ESs to form the dimeric synaptic complex, which catalyzes cleavage of the donor DNA. The transposome (dimeric transposase/DNA complex) then binds to the target DNA to transfer the transposon DNA to the target DNA.
Transposase-mediated NGS library preparation, depicted in Figure 1 , is similar to naturally- occurring transposition, except that the donor DNA or polynucleotide consists of two, separate adapter sequences rather than one continuous loop of donor DNA or polynucleotide. These adapter sequences are bound to transposases in a loading step that replaces the initial donor DNA or polynucleotide binding and cleavage step. Following adapter loading, the dimerized transposome proceeds to bind the target DNA or polynucleotide sample. Cleavage of the target polynucleotide and ligation of the adapter sequences results in fragmentation of the target DNA or polynucleotide and ligation of sequencing adapters to the target fragments.
The resulting DNA or polynucleotide fragments with ligated adapters comprise the next generation sequencing library. An ideal library preparation method would generate random fragments in the polynucleotide sequence, without bias toward the sequence composition of the target site. Compared to traditional library preparation methods, transposase-mediated fragmentation has lower sequence insertion bias. However, certain transposases are known to have sequence insertion bias. As an example, Tn5 has a slight G/C insertion bias. In some embodiments, the engineered transposases of the present disclosure have reduced sequence insertion bias or increased sequence promiscuity as compared to a wild-type or reference transposase.
In addition to containing the ME or ES sequences for target binding, adapters may comprise sequencing primers or other sequences used to identify or select fragments or groups of fragments from pooled samples. The adapters may encode primer binding sites for next generation sequencing library amplification, binding sites for sequencing primers, sample indexes, or other functional sequences related to a specific application. In some embodiments, the engineered transposases of the present disclosure have increased adapter loading or binding, as compared to a wild-type or reference transposase.
In some embodiments, further processing steps are used for library preparation, including gap filling, normalization, pooling of tagged fragment samples, and/or ligation of additional adapters to create dual-tagged libraries. A variety of parameters may be manipulated to create sequencing libraries with various characteristics. For example, varying the concentration of transposase and adapters can change the fragment length. Copy number and sample coverage may also be influenced or controlled using methods known to those of skill in the art.
As described further herein, the engineered transposase polypeptides of the current disclosure exhibit one of more improved properties in the transposase-mediated DNA fragmentation and adapter ligation process depicted in Figure 1.
In some embodiments, the present invention provides an engineered transposase polypeptide comprising an amino acid sequence having at least 60% sequence identity to an amino acid reference sequence of SEQ ID NO: 2 or SEQ ID NO: 5704 and further comprising one or more amino acid residue differences as compared to the reference amino acid sequence, wherein the engineered transposase polypeptide has increased polynucleotide fragmentation or cleavage activity, increased thermostability, increased activity at elevated temperatures, increased soluble expression, decreased inhibition, increased adapter loading or adapter ligation (i.e. tagging), increased binding to a target polynucleotide, and/or decreased sequence insertion bias as compared to another wild-type transposase or engineered transposase.
In particular, the engineered transposase polypeptides of the present disclosure have been engineered for efficient fragmentation or cleavage of DNA and the ligation of adapters in the process described above.
A variety of suitable reaction conditions are known to those skilled in the art, as detailed below and in the Examples.
Engineered Transposase Polypeptides
The present invention provides engineered transposase polypeptides useful in preparation of next generation sequencing libraries, as well as compositions and methods of utilizing these engineered polypeptides.
The present invention provides transposase polypeptides, polynucleotides encoding the polypeptides, methods of preparing the polypeptides, and methods for using the polypeptides. Where the description relates to polypeptides, it is to be understood that it can describe the polynucleotides encoding the polypeptides.
Suitable reaction conditions under which the above-described improved properties of the engineered polypeptides carry out the desired reaction can be determined with respect to concentrations or amounts of polypeptide, substrate, co-substrate, buffer, solvent, pH, conditions including temperature and reaction time, and/or conditions with the polypeptide immobilized on a solid support, as further described below and in the Examples.
In some embodiments, exemplary engineered transposases comprise an amino acid sequence that has one or more residue differences as compared to SEQ ID NO: 2 at the residue positions indicated in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 10.3, 10.5, 11.2, 11.4, 11.5, 12.2, 12.3, 12.5, 12.6, 13.2, 13.3, 13.4,
14.2, 14. 3, 14.5, 14.6, 14.8, 14.9, 15.1 , 16.2, 16.3, 17.2, 17.3, 18.2, 19.2, 19.3, 20.2, 20.3, 21.2, 21.3,
22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31 .2, 32.2, 33.2, 34.2, 35.1 . In some embodiments, exemplary engineered transposases comprise an amino acid sequence that has one or more residue differences as compared to SEQ ID NO: 5704 at the residue positions indicated in Tables 36.1 and 36.3.
The structure and function information for the exemplary engineered polypeptides of the present invention are based on the fragmentation or cleavage of double stranded DNA and the ligation or tagging of the DNA fragments, the results of which are shown below in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 10.3, 10.5, 11.2, 11.4, 11.5, 12.2, 12.3, 12.5, 12.6, 13.2, 13.3, 13.4, 14.2, 14. 3, 14.5, 14.6, 14.8, 14.9, 15.1 ,
16.2, 16.3, 17.2, 17.3, 18.2, 19.2, 19.3, 20.2, 20.3, 21 .2, 21 .3, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2,
29.2, 30.2, 31 .2, 32.2, 33.2, 34.2, 35.1 , 36.1 , and 36.3, as further described in the Examples. The odd numbered sequence identifiers (i.e., SEQ ID NOs) in these Tables refer to the nucleotide sequence encoding the amino acid sequence provided by the even numbered SEQ ID NOs in these Tables. Exemplary sequences are provided in the electronic sequence listing file accompanying this invention, which is hereby incorporated by reference herein. The amino acid residue differences are based on comparison to the reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, as indicated.
Proteins based on an IS4 transposase from Parashewanella curva or a Tn5 transposase with the ability to recognize the 19 bp Tn5 mosaic end (ME) transposon donor recognition sequence (SEQ ID NO: 2 or SEQ ID NO: 5704, respectively) were designed in silico, verified to have transposase activity, and selected for evolution. The transposase polypeptides of the present disclosure are engineered variants of SEQ ID NO: 2 or SEQ ID NO: 5704, which also comprise a C-terminal 6-histidine tag following a GGSG peptide spacer (SEQ ID NO: 5777).
The polypeptides of the present disclosure have residue differences that result in improved properties, including increased polynucleotide fragmentation or cleavage activity, increased thermostability, increased activity at elevated temperatures, increased soluble expression, decreased inhibition, increased adapter loading or adapter ligation (i.e. tagging), increased binding to a target polynucleotide, and/or decreased sequence insertion bias as compared to another wild-type transposase or engineered transposase. The activity of each engineered transposase relative to the reference polypeptide of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704 was determined as conversion of the substrates described in the Examples herein. In some embodiments, a shake flask purified enzyme (SFP) is used to assess the properties of the engineered transposases, the results of which are provided in the Examples.
In some embodiments, the specific enzyme properties are associated with the residues differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704 at the residue positions indicated herein. In some embodiments, residue differences affecting polypeptide expression can be used to increase expression of the engineered transposases.
In light of the guidance provided herein, it is further contemplated that any of the exemplary engineered polypeptides comprising the polypeptide sequences of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704 find use as the starting amino acid sequence for synthesizing other transposase polypeptides, for example by subsequent rounds of evolution that incorporate new combinations of various amino acid differences from other polypeptides in Tables 5.2, 6.2, 7.2, 8.2, 9.2,
10.2, 10.3, 10.5, 11.2, 11.4, 11.5, 12.2, 12.3, 12.5, 12.6, 13.2, 13.3, 13.4, 14.2, 14. 3, 14.5, 14.6, 14.8,
14.9, 15.1 , 16.2, 16.3, 17.2, 17.3, 18.2, 19.2, 19.3, 20.2, 20.3, 21.2, 21.3, 22.2, 23.2, 24.2, 25.2, 26.2,
27.2, 28.2, 29.2, 30.2, 31 .2, 32.2, 33.2, 34.2, 35.1 , 36.1 , and 36.3, and other residue positions described herein. Further improvements may be generated by including amino acid differences at residue positions that had been maintained as unchanged throughout earlier rounds of evolution.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2 and one or more residue differences as compared to SEQ ID NO: 2, selected from 10, 13, 24, 35, 39, 44, 46, 51 , 54, 55, 62/361 , 72, 75, 82, 89, 92, 107, 128, 137, 164, 167, 177/359, 191 , 192, 193, 194, 201 , 217, 281 , 289, 291 , 291/340, 305, 309, 310, 317, 331 , 337, 340, 346, 352, 353, 357, 358, 359, 362, 371 , 396, 414/435, 415, 428, 437, 443, 447, and 448. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2 and one or more residue differences as compared to SEQ ID NO: 2, selected from 10A, 13E, 24L, 35L, 39P, 44P, 46A, 51 E, 54T, 55K, 62D/361 H, 72L, 75H, 82R, 89V, 92L, 107G, 128A, 137L, 164E, 167R, 177I/359A, 191 Y, 192E, 193L, 194F, 201 G, 217G, 2811, 289P, 291 G, 291 Y/340L, 305V, 309E, 310D, 317W, 331 L, 337I, 340L, 346D, 352L, 353A, 357I, 358M, 358V, 359A, 362I, 371 L, 396I, 414D/435L, 415P, 428I, 437R, 443P, 447V, and 448L. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2 and one or more residue differences as compared to SEQ ID NO: 2, selected from S10A, H13E, R24L, M35L, S39P, T44P, S46A, K51 E, Q54T, E55K, R62D/R361 H, R72L, G75H, L82R, I89V, I92L, D107G, L128A, I137L, D164E, H167R, M177I/G359A, Q191Y, S192E, Y193L, L194F, N201 G, S217G, V281 I, K289P, D291 G, D291Y/Q340L, I305V, S309E, E310D, 1317W, W331 L, A337I, Q340L, R346D, V352L, S353A, F357I, I358M, I358V, G359A, L362I, P371 L, F396I, N414D/M435L, G415P, L428I, S437R, A443P, A447V, and I448L.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4 and one or more residue differences as compared to SEQ ID NO: 4, selected from 4, 5, 6, 8, 10, 10/317, 10/317/358, 10/317/359, 10/346, 10/346/359, 10/358, 10/359, 12, 12/373, 13, 15, 16, 18, 19, 26, 30, 30/154, 31 , 34, 35, 37, 38, 68, 70, 73, 74, 93, 106, 118, 126, 134, 135, 143, 146, 154/453, 163, 182, 207/286, 208, 226, 237, 246, 247, 259, 262, 263, 264, 280, 282, 286, 287, 288, 289, 291 , 292, 293, 298, 305, 309, 310, 313, 317, 317/346, 317/359, 317/445, 320, 346, 351 , 355, 356, 358, 359, 360, 368, 369, 410, 411 , 422, 426, 427, 428, 446, 448, 453, and 454. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4 and one or more residue differences as compared to SEQ ID NO: 4, selected from 4H, 5R, 6A, 8D, 10A, 10A/317W, 10A/317W/358V, 10A/317W/359A, 10A/346D, 10A/346D/359A, 10A/358V, 10A/359A, 12S, 12S/373C, 13V, 15S, 16S, 18A, 19L, 26A, 30N, 30N/154V, 31 V, 34Q, 35L, 37K, 38Y, 68A, 70A, 73K, 74A, 93E, 106E, 118S, 126V, 134F, 135R, 143E, 146M, 154P/453E, 163L, 182A, 207C/286I, 208S, 226K, 237S, 246K, 247R, 259S, 262L, 263R, 264S, 280A, 282L, 286I, 287N, 288P, 289P, 291 G, 292E, 293T, 298L, 305V, 309A, 310Q, 313R, 317W, 317W/346D, 317W/359A, 317W/445L, 320H, 346D, 351 M, 355L, 356S, 358V, 359A, 360V, 368S, 369F, 410G, 411 K, 422Q, 426M, 427A, 428I, 446G, 448L, 453E, and 454A. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4 and one or more residue differences as compared to SEQ ID NO: 4, selected from F4H, N5R, T6A, Q8D, S10A, S10A/I317W, S10A/I317W/I358V, S10A/I317W/G359A, S10A/R346D, S10A/R346D/G359A, S10A/I358V, S10A/G359A, E12S, E12S/Y373C, H13V, E15S, K16S, N18A, T19L, K26A, E30N, E30N/A154V, T31 V, D34Q, M35L, R37K, C38Y, P68A, V70A, C73K, S74A, D93E, S106E, A118S, T126V, T134F, K135R, D143E, C146M, A154P/D453E, A163L, S182A, R207C/T286I, A208S, E226K, D237S, S246K, K247R, K259S, V262L, K263R, V264S, V280A, Y282L, T286I, T287N, S288P, K289P, D291 G, M292E, E293T, V298L, I305V, S309A, E310Q, H313R, 1317W, I317W/R346D, I317W/G359A, I317W/W445L, A320H, R346D, A351 M, C355L, A356S, I358V, G359A, I360V, A368S, I369F, P410G, R411 K, K422Q, Q426M, S427A, L428I, E446G, I448L, D453E, and T454A.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 156 and one or more residue differences as compared to SEQ ID NO: 156, selected from 18/73/74/259, 18/73/286/289/411 , 18/74/118/259/286/289/411 , 18/74/259, 18/74/259/286/289, 18/74/259/286/411 , 18/118/259, 18/259/286/289/411 , 18/259/289/411 , 18/289/411 , 73/74/259/286/289, 73/74/259/289, 73/259/286, 74/259, 74/259/289, 74/286/289/411 , 259, 259/286/289, and 289. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 156 and one or more residue differences as compared to SEQ ID NO: 156, selected from 18A/73K/74A/259S, 18A/73K/286I/289P/411 K, 18A/74A/118S/259S/286I/289P/411 K, 18A/74A/259S,
18A/74A/259S/286I/289P, 18A/74A/259S/286I/411 K, 18A/118S/259S, 18A/259S/286I/289P/411 K, 18A/259S/289P/411 K, 18A/289P/411 K, 73K/74A/259S/286I/289P, 73K/74A/259S/289P, 73K/259S/286I, 74A/259S, 74A/259S/289P, 74A/286I/289P/411 K, 259S, 259S/286I/289P, and 289P. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 156 and one or more residue differences as compared to SEQ ID NO: 156, selected from N18A/C73K/S74A/K259S,
N18A/C73K/T286I/K289P/R411 K, N18A/S74A/A118S/K259S/T286I/K289P/R411 K, N18A/S74A/K259S, N18A/S74A/K259S/T286I/K289P, N18A/S74A/K259S/T286I/R411 K, N18A/A118S/K259S, N18A/K259S/T286I/K289P/R411 K, N18A/K259S/K289P/R411 K, N18A/K289P/R411 K, C73K/S74A/K259S/T286I/K289P, C73K/S74A/K259S/K289P, C73K/K259S/T286I, S74A/K259S, S74A/K259S/K289P, S74A/T286I/K289P/R411 K, K259S, K259S/T286I/K289P, and K289P.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 302 and one or more residue differences as compared to SEQ ID NO: 302, selected from 16/19/355/427, 16/37/146/286/287/289/310/313/355, 16/146/226/287/289/355, 19/37/146/286/287/310/355, 19/135/286/287/289/355, 19/182/355, 37/70/226/310/313/355, 37/74/310/355/427, 37/135/146/182/226/286/287/355/368, 37/146/226/289/427, 37/146/287/289/427, 38, 41 , 42, 43, 44, 45, 47, 48, 50, 53, 54, 55, 70/74/226/286/287/289/368/427, 70/74/355/368, 70/135/146/150/182/286/289/313/427, 74/310/313/355, 74/310/355, 74/355/427, 101 , 103, 117, 135/226/286/289/355/368, 135/355, 146, 146/150/287/289/310/355, 146/182/355, 146/226/286/355, 146/286/287/289/355/427, 146/289, 146/427, 150/226/310/313, 182/226/355, 182/355, 244, 247, 249, 251 , 287/355/427, 300, 317/411 , 355, 393, 397, 398, 399, 401 , 405, 407, 408, 409, 411 , 413, 426, 429, 431 , 439, 444, 446, 450, 453, 454, 457, 459, 460, 464, 466, 467, 469, 470, 471 , 472, 474, 475, 476, and 478. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 302 and one or more residue differences as compared to SEQ ID NO: 302, selected from 16S/19L/355L/427A, 16S/37K/146M/286I/287N/289P/31 OQ/313R/355L, 16S/146M/226K/287N/289P/355L, 19L/37K/146M/286I/287N/310Q/355L, 19L/135R/286I/287N/289P/355L, 19L/182A/355L, 37K/70A/226K/31 OQ/313R/355L, 37K/74S/310Q/355L/427A, 37K/135R/146M/182A/226K/286I/287N/355L/368S, 37K/146M/226K/289P/427A, 37K/146M/287N/289P/427A, 38F, 38N, 41 R, 42G, 43L, 43V, 44G, 44S, 44V, 45M, 45V, 47M, 47S, 48D, 50D, 53D, 53L, 54H, 54S, 54V, 55M, 70A/74S/226K/286I/287N/289P/368S/427A, 70A/74S/355L/368S, 70A/135R/146M/150H/182A/286I/289P/313R/427A, 74S/31 OQ/313R/355L, 74S/310Q/355L, 74S/355L/427A, 101 S, 103M, 103R, 117R, 135R/226K/286I/289P/355L/368S, 135R/355L, 146M, 146M/150H/287N/289P/310Q/355L, 146M/182A/355L, 146M/226K/286I/355L, 146M/286I/287N/289P/355L/427A, 146M/289P, 146M/427A, 150H/226K/31 OQ/313R, 182A/226K/355L, 182A/355L, 244I, 244T, 247L, 249F, 249I, 249L, 249S, 249T, 249V, 249Y, 251 L, 287N/355L/427A, 300F, 317R/411 N, 355L, 393A, 397S, 398V, 399V, 401 V, 405F, 405V, 407L, 408R, 409G, 411 D, 413Y, 426A, 426H, 426M, 426S, 426V, 426W, 429G, 431 M, 439G, 444G, 446K, 446Q, 450L, 450M, 450T, 453G, 453H, 453R, 453T, 454L, 454R, 457A, 459M, 460T, 464L, 464M, 466R, 467G, 469F, 470M, 470N, 470R, 470V, 470Y, 471 A, 471 S, 472R, 474R, 474S, 475P, 476D, 476L, 476S, 476T, 478C, 478G, 478L, and 478V. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 302 and one or more residue differences as compared to SEQ ID NO: 302, selected from K16S/T19L/C355L/S427A, K16S/R37K/C146M/T286I/T287N/K289P/E310Q/H313R/C355L,
K16S/C146M/E226K/T287N/K289P/C355L, T19L/R37K/C146M/T286I/T287N/E310Q/C355L, T19L/K135R/T286I/T287N/K289P/C355L, T19L/S182A/C355L, R37K/V70A/E226K/E310Q/H313R/C355L, R37K/A74S/E310Q/C355L/S427A, R37K/K135R/C146M/S182A/E226K/T286I/T287N/C355L/A368S, R37K/C146M/E226K/K289P/S427A, R37K/C146M/T287N/K289P/S427A, C38F, C38N, K41 R, S42G, I43L, I43V, T44G, T44S, T44V, I45M, I45V, C47M, C47S, Q48D, S50D, M53D, M53L, Q54H, Q54S, Q54V, E55M, V70A/A74S/E226K/T286I/T287N/K289P/A368S/S427A, V70A/A74S/C355L/A368S, V70A/K135R/C146M/N150H/S182A/T286I/K289P/H313R/S427A, A74S/E310Q/H313R/C355L, A74S/E310Q/C355L, A74S/C355L/S427A, K101 S, Q103M, Q103R, K117R, K135R/E226K/T286I/K289P/C355L/A368S, K135R/C355L, C146M,
C146M/N150H/T287N/K289P/E310Q/C355L, C146M/S182A/C355L, C146M/E226K/T286I/C355L, C146M/T286I/T287N/K289P/C355L/S427A, C146M/K289P, C146M/S427A, N150H/E226K/E310Q/H313R, S182A/E226K/C355L, S182A/C355L, V244I, V244T, K247L, K249F, K249I, K249L, K249S, K249T, K249V, K249Y, K251 L, T287N/C355L/S427A, L300F, W317R/R411 N, C355L, E393A, D397S, E398V, D399V, W401 V, M405F, M405V, M407L, K408R, K409G, R411 D, W413Y, Q426A, Q426H, Q426M, Q426S, Q426V, Q426W, A429G, L431 M, R439G, S444G, E446K, E446Q, E450L, E450M, E450T, D453G, D453H, D453R, D453T, T454L, T454R, S457A, V459M, V460T, V464L, V464M, K466R, E467G, L469F, A470M, A470N, A470R, A470V, A470Y, D471 A, D471 S, G472R, L474R, L474S, L475P, G476D, G476L, G476S, G476T, S478C, S478G, S478L, and S478V.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 338 and one or more residue differences as compared to SEQ ID NO: 338, selected from 16, 16/313, 16/409/411 /427, 37/150/408, 37/313/317/408/411 , 37/317/405/408, 37/317/405/426, 37/405/409/427, 150, 150/313/405/411 , 150/405/406, 150/408, 150/408/411 , 226/405, 317/460, and 408/426. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 338 and one or more residue differences as compared to SEQ ID NO: 338, selected from 16S, 16S/313R, 16S/409D/411 N/427M, 37K/150N/408G, 37K/313R/317R/408G/411 N, 37K/317R/405V/408G,
37K/317R/405V/426M, 37K/405V/409D/427M, 150N, 150N/313R/405V/411 N, 150N/405V/406S, 150N/408G, 150N/408G/411 N, 226K/405V, 317R/460L, and 408G/426M. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 338 and one or more residue differences as compared to SEQ ID NO: 338, selected from K16S, K16S/H313R, K16S/K409D/R411 N/S427M, R37K/H150N/K408G, R37K/H313R/W317R/K408G/R411 N, R37K/W317R/M405V/K408G, R37K/W317R/M405V/Q426M, R37K/M405V/K409D/S427M, H150N, H150N/H313R/M405V/R411 N, H150N/M405V/K406S, H150N/K408G, H150N/K408G/R411 N, E226K/M405V, W317R/V460L, and K408G/Q426M.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from 12, 13, 18, 19, 22, 26, 317262, 34, 60, 79, 80, 81 , 82, 92, 106, 107, 113, 115, 132, 134, 136,
140, 143, 152, 160, 163, 167, 168, 175, 179, 183, 195, 199, 201 , 202, 215, 216, 218, 220, 223, 226, 233,
235, 236, 237, 243, 255, 259, 261 , 262, 266, 268, 270, 273, 282, 290, 292, 293, 309, 319, 320, 326, 343,
346, 365, 370, 374, 380, 382, 386, 389, 390, 408, 412, 414, 415, 424, and 442. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from 12M, 131, 18Q, 19L, 22S, 22T, 26R, 31 M/262A, 34G, 60L, 79Y, 80T, 81 T, 82Q, 92L, 92T, 106Q, 107G, 107H, 107R, 113K, 115R, 132G, 132R, 134E, 134G, 136L, 140C, 140L, 140V, 143G, 143R, 143V, 152S, 160Q, 160T, 163L, 163T, 167R, 168Q, 175T, 179G, 183L, 195G, 199R, 199T, 199W, 201 G, 201 Q, 202M, 215R, 215V, 216E, 218G, 220R, 223R, 223V, 226L, 226Q, 233R, 235V, 236T, 237G, 237S, 243L, 243M, 255R, 259A, 261 E, 262L, 266P, 268A, 270R, 273K, 273P, 273T, 282G, 290A, 290R, 290T, 292W, 293V, 309G, 319R, 320R, 320S, 326A, 326L, 343G, 343R, 346G, 346N, 365I, 370F, 374L, 380R, 382A, 382R, 386D, 389G, 390R, 408A, 408R, 412A, 412H, 4121, 412R, 414R, 415S, 424V, and 442K. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from E12M, H13I, A18Q, T19L, P22S, P22T, K26R, T31 M/V262A, D34G, F60L, T79Y, A80T, V81T, L82Q, I92L, I92T, S106Q, D107G, D107H, D107R, S113K, K115R, F132G, F132R, T134E, T134G, T136L, I140C, I140L, 1140V, D143G, D143R, D143V, E152S, G160Q, G160T, A163L, A163T, H167R, F168Q, D175T, R179G, V183L, Q195G, S199R, S199T, S199W, N201 G, N201 Q, E202M, L215R, L215V, D216E, E218G, P220R, E223R, E223V, E226L, E226Q, A233R, T235V, I236T, D237G, D237S, V243L, V243M, A255R, S259A, S261 E, V262L, A266P, T268A, K270R, D273K, D273P, D273T, Y282G, K290A, K290R, K290T, M292W, E293V, S309G, T319R, A320R, A320S, E326A, E326L, E343G, E343R, R346G, R346N, L365I, T370F, Y374L, L380R, D382A, D382R, E386D, S389G, Q390R, G408A, G408R, G412A, G412H, G412I, G412R, N414R, G415S, A424V, and I442K.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from 2, 12, 13, 15, 16, 18, 19, 22, 26, 30, 31/262, 34, 51 , 60, 69, 79, 82, 84, 87, 88, 106, 107, 113, 114, 115, 128, 132, 134, 135, 136, 140, 143, 146, 150, 152, 160, 163, 167, 168, 175, 179, 183, 195,
198, 199, 201 , 215, 217, 218, 219, 220, 223, 224, 226, 227, 231 , 233, 235, 236, 243, 254, 255, 259, 261 ,
266, 267, 268, 270, 272, 273, 275, 277, 279, 282, 287, 289, 290, 291 , 292, 293, 304, 307, 309, 319, 320,
343, 344, 346, 355, 365, 370, 373, 380, 382, 386, 389, 390, 408, 412, 414, 415, 418, 423, 424, and 468.
In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from 2A, 12L, 12M, 131, 13N, 13Y, 15R, 16S, 18Q, 19L, 22S, 22T, 26R, 30H, 31 M/262A, 34G, 51 R, 60L, 69A, 69L, 79Y, 82Q, 84R, 87T, 88L, 106Q, 107G, 107H, 107R, 113E, 114P, 115S, 128F, 132G, 132R, 134E, 134G, 134L, 135T, 136L, 140C, 140L, 140V, 143G, 143R, 143V, 146K, 150R, 152S, 160Q, 160T, 163L, 163T, 167R, 168Q, 175T, 175V, 179G, 183L, 195G, 195S, 198S, 199R, 199T, 199W, 201 G, 201 Q, 215P, 215V, 217F, 217G, 218G, 219S, 220R, 223R, 223V, 224A, 224S, 226L, 227Y, 231 V, 233R, 235A, 235V, 236T, 243L, 243M, 254F, 255R, 259A, 261 E, 266P, 267P, 268A, 270A, 270R, 272L, 272P, 273K, 273P, 273T, 275L, 277K, 279G, 282G, 287L, 287R, 289T, 290T, 291 S, 292W, 293V, 304R, 307L, 309G, 319R, 320L, 320N, 320R, 320S, 343G, 343R, 344V, 346G, 346N, 355G, 365I, 370F, 373R, 380G, 380R, 382R, 386D, 386Q, 389G, 390R, 408A, 412A, 412H, 4121, 412R, 414R, 415S, 418S, 423D, 424V, and 468T. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from S2A, E12L, E12M, H13I, H13N, H13Y, E15R, K16S, A18Q, T19L, P22S, P22T, K26R, E30H, T31 M/V262A, D34G, K51 R, F60L, E69A, E69L, T79Y, L82Q, Q84R, P87T, E88L, S106Q, D107G, D107H, D107R, S113E, V114P, K115S, L128F, F132G, F132R, T134E, T134G, T134L, K135T, T136L, I140C, I140L, 1140V, D143G, D143R, D143V, M146K, H150R, E152S, G160Q, G160T, A163L, A163T, H167R, F168Q, D175T, D175V, R179G, V183L, Q195G, Q195S, Q198S, S199R, S199T, S199W, N201 G, N201 Q, L215P, L215V, S217F, S217G, E218G, L219S, P220R, E223R, E223V, Y224A, Y224S, E226L, S227Y, L231 V, A233R, T235A, T235V, I236T, V243L, V243M, P254F, A255R, S259A, S261 E, A266P, V267P, T268A, K270A, K270R, K272L, K272P, D273K, D273P, D273T, M275L, P277K, N279G, Y282G, N287L, N287R, P289T, K290T, D291 S, M292W, E293V, P304R, T307L, S309G, T319R, A320L, A320N, A320R, A320S, E343G, E343R, E344V, R346G, R346N, L355G, L365I, T370F, Y373R, L380G, L380R, D382R, E386D, E386Q, S389G, Q390R, G408A, G412A, G412H, G412I, G412R, N414R, G415S, V418S, W423D, A424V, and M468T.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from 44/45/47/408/453, 44/45/47/453, 44/45/47/453/454, 44/45/103/453/457, 44/45/453/454/457, 44/47/103/393/408/453/454, 44/47/103/453/454/457, 44/47/393/408/453/457, 44/47/393/453/457, 44/103/408/453/454, 45/47/103/393/453/454/457, 45/47/393/453/454, 45/47/408/423/453/457, 45/47/453, 45/103/393/453/454, 45/453/454, 47/103/408/453/454, 47/393/408/453/454/457, 47/393/453/454, 47/408/453/457, 53/54/244/450, 103/393/408/453/457, 244/450, 408/453/454, 446/450/464, and 453/457. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from 44G/45V/47M/408R/453R, 44G/45V/47M/453H, 44G/45V/47M/453R/454R, 44G/47M/393A/453R/457G, 44S/45V/103M/453R/457G, 44S/45V/453T/454R/457G, 44S/47M/103M/393A/408R/453R/454R, 44S/47M/103M/453R/454R/457G, 44S/47S/393A/408R/453R/457G, 44S/103M/408R/453R/454R, 45V/47M/393A/453R/454R, 45V/47M/408R/423C/453T/457G, 45V/47S/103M/393A/453R/454R/457G, 45V/47S/453R, 45V/103M/393A/453T/454R, 45 V/453 R/454R, 47M/103M/408R/453T/454R, 47M/393A/408R/453R/454R/457G, 47M/393A/453R/454R, 47S/408R/453R/457G, 53D/54V/244I/450T, 103M/393A/408R/453R/457G, 244I/450T, 408R/453T/454R, 446K/450T/464L, and 453R/457G. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 606 and one or more residue differences as compared to SEQ ID NO: 606, selected from T44G/I45V/C47M/G408R/D453R, T44G/I45V/C47M/D453H, T44G/I45V/C47M/D453R/T454R, T44G/C47M/E393A/D453R/S457G, T44S/I45V/Q103M/D453R/S457G, T44S/I45V/D453T/T454R/S457G, T44S/C47M/Q103M/E393A/G408R/D453R/T454R, T44S/C47M/Q103M/D453R/T454R/S457G, T44S/C47S/E393A/G408R/D453R/S457G, T44S/Q103M/G408R/D453R/T454R, I45V/C47M/E393A/D453R/T454R, I45V/C47M/G408R/W423C/D453T/S457G, I45V/C47S/Q103M/E393A/D453R/T454R/S457G, I45V/C47S/D453R, I45V/Q103M/E393A/D453T/T454R, I45V/D453R/T454R, C47M/Q103M/G408R/D453T/T454R, C47M/E393A/G408R/D453R/T454R/S457G, C47M/E393A/D453R/T454R, C47S/G408R/D453R/S457G, M53D/Q54V/V244I/E450T, Q103M/E393A/G408R/D453R/S457G, V244I/E450T, G408R/D453T/T454R, E446K/E450T/V464L, and D453R/S457G.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from 38/45/53, 38/45/53/54/476, 38/45/53/244, 38/45/53/470/472, 38/45/54/244, 38/45/54/470/474/478, 38/45/472/476/478, 38/45/476, 38/53/54, 38/53/54/244, 38/53/54/244/472/478, 38/53/472, 38/54/244/470/474/476/478, 38/244/470/472/474/478, 38/478, 45/53/54/244/470/474/476/478, 45/53/54/470/474/476, 45/53/54/472/478, 45/54/244/474/478, 53, 53/54/470/476/478, 53/244/474/478, 54, 54/244, 54/244/472/476, 54/472, 244, and 474/478. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from 38F/45I/53D, 38F/45I/53D/54S/476S, 38F/45I/53L/244I, 38F/45I/53L/470N/472R, 38F/45I/54V/244T, 38F/45I/54V/470N/474S/478G, 38F/45I/472R/476S/478G, 38F/45I/476S, 38F/53D/54H/244I/472R/478G, 38F/53D/54S, 38F/53D/54S/244I, 38F/53D/54S/244I/472R/478G, 38F/53L/472R, 38F/54S/244I/470N/474S/476S/478G, 38F/244I/470R/472R/474S/478G, 38F/478G, 451/53 D/54H/244I/470N/474S/476S/478G , 451/53 D/54S/470 R/474S/476S , 451/53L/54S/472 R/478G , 45I/54S/244T/474S/478G, 53D, 53D/244T/474S/478G, 53L/54S/470R/476S/478G, 54H, 54S/472R, 54V/244I/472R/476S, 54V/244T, 244T, and 474S/478G. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from C38F/V45I/M53D, C38F/V45I/M53D/Q54S/G476S, C38F/V45I/M53L/V244I, C38F/V45I/M53L/A470N/G472R, C38F/V45I/Q54V/V244T, C38F/V45I/Q54V/A470N/L474S/S478G, C38F/V45I/G472R/G476S/S478G, C38F/V45I/G476S, C38F/M53D/Q54H/V244I/G472R/S478G, C38F/M53D/Q54S, C38F/M53D/Q54S/V244I, C38F/M53D/Q54S/V244I/G472R/S478G, C38F/M53L/G472R, C38F/Q54S/V244I/A470N/L474S/G476S/S478G, C38 F/V244I/A470 R/G472 R/L474S/S478G , C38 F/S478G ,
V45I/M53D/Q54H/V244I/A470N/L474S/G476S/S478G, V45I/M53D/Q54S/A470R/L474S/G476S, V45I/M53L/Q54S/G472R/S478G, V45I/Q54S/V244T/L474S/S478G, M53D, M53D/V244T/L474S/S478G, M53L/Q54S/A470R/G476S/S478G, Q54H, Q54S/G472R, Q54V/V244I/G472R/G476S, Q54V/V244T, V244T, and L474S/S478G.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from 22/38/45/107/259/317/412, 22/38/45/259/317/412, 22/107, 22/107/317/412, 22/134, 22/134/220, 22/134/220/317, 22/134/317/412, 22/317, 38, 38/45/81/143/244, 38/53/243, 38/81 , 38/81/244, 38/107/134/220, 38/132, 38/220/317, 45/81 , 45/81/243, 45/81/243/343, 45/143/243/270/343, 45/244/270, 53/115/163/243/270/343, 53/143/243/270/343, 53/270, 64/134/220/317/412, 81/132/243, 107, 107/134/317, 115/244/270, 134/259, 143/243/270, 243, 243/270, 244, 244/270, 259, 270, and 317. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from 22T/38F/45I/107R/259A/317M/412H, 22T/38F/45I/259A/317M/412H, 22T/107R, 22T/107R/317M/412H, 22T/134G, 22T/134G/220R/317M, 22T/134G/317M/412H, 22T/134L/220R, 22T/317M, 38F, 38F/45I/81 T/143R/244I, 38F/53D/243L, 38F/81T, 38F/81 T/244I, 38F/107R/134G/220R, 38F/132G, 38F/220R/317M, 451/81 T, 451/81 T/243L, 451/81 T/243L/343R, 451/143R/243L/270R/343R, 45I/244I/270R, 53D/115R/163L/243L/270R/343R, 53D/143R/243L/270R/343R, 53D/270R, 64R/134L/220R/317M/412H, 81T/132G/243L, 107R, 107R/134L/317M, 115R/244I/270R, 134G/259A, 143R/243L/270R, 243L, 243L/270R, 244I, 244I/270R, 259A, 270R, and 317M. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from
P22T/C38F/V45I/D107R/S259A/R317M/G412H, P22T/C38F/V45I/S259A/R317M/G412H, P22T/D107R, P22T/D107R/R317M/G412H, P22T/T134G, P22T/T134G/P220R/R317M, P22T/T134G/R317M/G412H, P22T/T134L/P220R, P22T/R317M, C38F, C38F/V45I/V81T/D143R/V244I, C38F/M53D/V243L, C38F/V81T, C38F/V81T/V244I, C38F/D107R/T134G/P220R, C38F/F132G, C38F/P220R/R317M, V45I/V81T, V45I/V81T/V243L, V45I/V81T/V243L/E343R, V45I/D143R/V243L/K270R/E343R, V45I/V244I/K270R, M53D/K115R/A163L/V243L/K270R/E343R, M53D/D143R/V243L/K270R/E343R, M53D/K270R, P64R/T134L/P220R/R317M/G412H, V81T/F132G/V243L, D107R, D107R/T134L/R317M, K115R/V244I/K270R, T134G/S259A, D143R/V243L/K270R, V243L, V243L/K270R, V244I, V244I/K270R, S259A, K270R, and R317M.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from 22/38/45/107/259/317/412, 22/38/45/259/317/412, 22/38/64/134/220, 22/45/220/259, 22/64/107/259/317/412, 22/64/134/220, 22/107, 22/134, 22/134/317, 22/134/317/412, 22/220, 22/220/412/415, 22/259/317, 22/259/317/412, 22/317, 38, 38/45/81/143/244, 38/45/143/270, 38/53/243, 38/81 , 38/81/143/243/270, 38/81/183/243/270, 38/81/243, 38/81/244, 38/107/134/220, 38/132, 38/220/317, 38/270/343, 45/81 , 45/81/132/243/270, 45/81/243, 45/143/243/270/343, 45/244/270, 53/143/243/270/343, 53/244/270, 53/270, 64, 64/107/134, 64/107/317, 64/134, 64/134/220/317/412, 81/132, 81/132/243, 107, 107/134/317, 107/412, 115/244/270, 143/243/270, 220/259, 243, 243/270, 244/270, 255, 259, and 270. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from 22T/38F/45I/107R/259A/317M/412H, 22T/38F/45I/259A/317M/412H, 22T/38F/64R/134L/220R, 22T/45I/220R/259A, 22T/64R/107R/259A/317M/412H, 22T/64R/134L/220R, 22T/107R, 22T/134G, 22T/134G/317M/412H, 22T/134L/317M, 22T/134L/317M/412H, 22T/220R, 22T/220R/412H/415D, 22T/259A/317M, 22T/259A/317M/412H, 22T/317M, 38F, 38F/45I/81 T/143R/244I, 38F/45I/143R/270R, 38F/53D/243L, 38F/81T, 38F/81T/143R/243L/270R, 38F/81T/183L/243L/270R, 38F/81T/243L, 38F/81 T/244I, 38F/107R/134G/220R, 38F/132G, 38F/220R/317M, 38F/270R/343R, 451/81 T, 451/81 T/132G/243L/270R, 451/81 T/243L, 451/143R/243L/270R/343R, 45I/244I/270R, 53D/143R/243L/270R/343R, 53D/244I/270R, 53D/270R, 64R, 64R/107R/134L, 64R/107R/317M, 64R/134L, 64R/134L/220R/317M/412H, 81T/132G, 81 T/132G/243L, 107R, 107R/134L/317M, 107R/412H, 115R/244I/270R, 143R/243L/270R, 220R/259A, 243L, 243L/270R, 244I/270R, 255R, 259A, and 270R. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from
P22T/C38F/V45I/D107R/S259A/R317M/G412H, P22T/C38F/V45I/S259A/R317M/G412H, P22T/C38F/P64R/T134L/P220R, P22T/V45I/P220R/S259A, P22T/P64R/D107R/S259A/R317M/G412H, P22T/P64R/T134L/P220R, P22T/D107R, P22T/T134G, P22T/T134G/R317M/G412H, P22T/T134L/R317M, P22T/T134L/R317M/G412H, P22T/P220R, P22T/P220R/G412H/G415D, P22T/S259A/R317M, P22T/S259A/R317M/G412H, P22T/R317M, C38F, C38F/V45I/V81T/D143R/V244I, C38F/V45I/D143R/K270R, C38F/M53D/V243L, C38F/V81T, C38F/V81T/D143R/V243L/K270R, C38F/V81T/V183L/V243L/K270R, C38F/V81T/V243L, C38F/V81T/V244I, C38F/D107R/T134G/P220R, C38F/F132G, C38F/P220R/R317M, C38F/K270R/E343R, V45I/V81T, V45I/V81T/F132G/V243L/K270R, V45I/V81 T/V243L, V45I/D143R/V243L/K270R/E343R, V45I/V244I/K270R, M53D/D143R/V243L/K270R/E343R, M53D/V244I/K270R, M53D/K270R, P64R, P64R/D107R/T134L, P64R/D107R/R317M, P64R/T134L, P64R/T134L/P220R/R317M/G412H, V81T/F132G, V81T/F132G/V243L, D107R, D107R/T134L/R317M, D107R/G412H, K115R/V244I/K270R, D143R/V243L/K270R, P220R/S259A, V243L, V243L/K270R, V244I/K270R, A255R, S259A, and K270R.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 143, 143/175/272/273/415, 152/168/216/237, 152/168/423, 160, 160/201/272, 160/201/273/415, 160/226, 160/244/272/273/415, 160/415, 168, 168/187/216/255/423, 168/216/237, 168/255/423, 168/290/423, 187, 187/423, 199/201/226, 199/201/415, 201 , 216, 216/423, 226/244/415, 226/415, 237, 237/290, 244, 255, 272, 290, 290/423, 415, and 423. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 143R, 143R/175T/272W/273P/415S, 152S/168Q/216E/237S, 152S/168Q/423D, 160Q, 160Q/201 G/272W, 160Q/201 G/273P/415S, 160Q/226R, 160Q/244I/272W/273P/415S, 160Q/415S, 168Q, 168Q/187G/216E/255R/423D, 168Q/216E/237S, 168Q/255R/423D, 168Q/290R/423D, 187G, 187G/423D, 199W/201 G/226R, 199W/201 G/415S, 201 G, 216E, 216E/423D, 226R/244I/415S, 226R/415S, 237S, 237S/290R, 244I, 255R, 272W, 290R, 290R/423D, 415S, and 423D. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from D143R, D143R/D175T/K272W/D273P/G415S, E152S/F168Q/D216E/D237S, E152S/F168Q/W423D, G160Q, G160Q/N201 G/K272W, G160Q/N201 G/D273P/G415S, G160Q/E226R, G160Q/V244I/K272W/D273P/G415S, G160Q/G415S, F168Q, F168Q/E187G/D216E/A255R/W423D, F168Q/D216E/D237S, F168Q/A255R/W423D, F168Q/K290R/W423D, E187G, E187G/W423D, S199W/N201 G/E226R, S199W/N201 G/G415S, N201 G, D216E, D216E/W423D, E226R/V244I/G415S, E226R/G415S, D237S, D237S/K290R, V244I, A255R, K272W, K290R, K290R/W423D, G415S, and W423D.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 143, 143/160/226/415, 143/175/272/273/415, 152/168/216/237, 152/168/423, 160, 160/201/272, 160/201/273/415, 160/226, 160/244/272/273/415, 160/415, 168, 168/187/216/255/423, 168/216/237, 168/216/237/255/343, 168/255/423, 168/290/423, 187, 199/201/226, 199/201/415, 201 , 216, 216/423, 226/244/415, 226/415, 237, 237/290, 244, 255, 255/343, 272, 290, 290/423, 415, and 423. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 143R, 143R/160T/226R/415S, 143R/175T/272W/273P/415S, 152S/168Q/216E/237S, 152S/168Q/423D, 160Q, 160Q/201 G/272W, 160Q/201 G/273P/415S, 160Q/226R, 160Q/244I/272W/273P/415S, 160Q/415S, 168Q, 168Q/187G/216E/255R/423D, 168Q/216E/237S, 168Q/216E/237S/255R/343R, 168Q/255R/423D, 168Q/290R/423D, 187G, 199W/201 G/226R, 199W/201 G/415S, 201 G, 216E, 216E/423D, 226R/244I/415S, 226R/415S, 237S, 237S/290R, 244I, 255R, 255R/343R, 272W, 290R, 290R/423D, 415S, and 423D. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from D143R, D143R/G160T/E226R/G415S, D143R/D175T/K272W/D273P/G415S, E152S/F168Q/D216E/D237S, E152S/F168Q/W423D, G160Q, G160Q/N201 G/K272W, G160Q/N201 G/D273P/G415S, G160Q/E226R, G160Q/V244I/K272W/D273P/G415S, G160Q/G415S, F168Q, F168Q/E187G/D216E/A255R/W423D, F168Q/D216E/D237S, F168Q/D216E/D237S/A255R/E343R, F168Q/A255R/W423D, F168Q/K290R/W423D, E187G, S199W/N201 G/E226R, S199W/N201 G/G415S, N201 G, D216E, D216E/W423D, E226R/V244I/G415S, E226R/G415S, D237S, D237S/K290R, V244I, A255R, A255R/E343R, K272W, K290R, K290R/W423D, G415S, and W423D.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 72, 90, 91 , 92, 93, 95, 122, 129, 141 , 142, 143, 144, 145, 150, 151 , 151/155, 152, 154, 155, 157, 160, 161 , 161/313, 164, 165, 168, 180, 181 , 184, 193, 197, 204, 205, 205/472, 206, 299, and 320. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 72M, 72Q, 90F, 911, 91 V, 92A, 92L, 92V, 93A, 93H, 93S, 95G, 95S, 122F, 122L, 122Q, 122V, 129F, 1291, 141 Y, 142L, 143A, 143K, 144F, 1441, 144V, 144Y, 145F, 145L, 150D, 150E, 150G, 150R, 150S, 150T, 150V, 150W, 151 A, 151Q, 151 R, 151T/155T, 152D, 152H, 152L, 152Q, 152R, 152T, 152V, 152Y, 154E, 154K, 154L, 154P, 154Q, 154S, 154T, 154V, 154Y, 155E, 155G, 155H, 155Q, 155R, 155S, 155T, 157R, 1601, 160Q, 160V, 160W, 161 C, 161 C/313C, 161 R, 161 V, 164Q, 164R, 164S, 165G, 165S, 168E, 168K, 168L, 168N, 168R, 168V, 180C, 180L, 180T, 181 L, 181 V, 184A, 184G, 184H, 184S, 193H, 193L, 193M, 193V, 197H, 197R, 197Y, 204L, 204M, 204V, 204W, 204Y, 205I, 205L, 205L/472D, 206L, 299F, 299I, 320C, 320H, 320K, 320L, 320Q, 320R, 320S, and 320Y. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from R72M, R72Q, L90F, A911, A91 V, I92A, I92L, I92V, D93A, D93H, D93S, T95G, T95S, W122F, W122L, W122Q, W122V, L129F, L129I, H141Y, Q142L, D143A, D143K, W144F, W144I, W144V, W144Y, W145F, W145L, H150D, H150E, H150G, H150R, H150S, H150T, H150V, H150W, P151 A, P151 Q, P151 R, P151T/D155T, E152D, E152H, E152L, E152Q, E152R, E152T, E152V, E152Y, A154E, A154K, A154L, A154P, A154Q, A154S, A154T, A154V, A154Y, D155E, D155G, D155H, D155Q, D155R, D155S, D155T, K157R, G160I, G160Q, G160V, G160W, K161 C, K161 C/R313C, K161 R, K161 V, D164Q, D164R, D164S, A165G, A165S, F168E, F168K, F168L, F168N, F168R, F168V, V180C, V180L, V180T, 1181 L, 1181 V, C184A, C184G, C184H, C184S, Y193H, Y193L, Y193M, Y193V, K197H, K197R, K197Y, F204L, F204M, F204V, F204W, F204Y, V205I, V205L, V205L/G472D, V206L, L299F, L299I, A320C, A320H, A320K, A320L, A320Q, A320R, A320S, and A320Y.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 90, 92, 122, 129, 141 , 143, 144, 145, 150, 151 , 151/155, 152, 154, 155, 155/265, 157, 160, 161 , 164, 165, 168, 180, 181 , 184, 193, 197, 204, 205, 205/472, 206, 299, and 320. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from 90F, 92A, 92L, 92P, 92V, 122F, 122L, 129F, 1291, 141 Y, 143A, 143K, 144F, 144Y, 145F, 145L, 150D, 150E, 150G, 150R, 150S, 150V, 150W, 151 A, 151 K, 151Q, 151 R, 151T/155T, 152D, 152H, 152L, 152Q, 152R, 152T, 152V, 152Y, 154E, 154K, 154L, 154Q, 154S, 154T, 154V, 154Y, 155E, 155G, 155H, 155Q, 155Q/265V, 155R, 155S, 155T, 157R, 1601, 160Q, 160V, 160W, 161 C, 161 R, 161 V, 164Q, 164R, 164S, 165G, 165S, 168E, 168K, 168L, 168N, 168R, 168V, 180A, 180C, 180L, 180T, 181 L, 181 V, 184A, 184G, 184S, 193H, 193L, 193M, 193V, 197H, 197R, 204L, 204M, 204V, 204W, 204Y, 205I, 205L, 205L/472D, 206L, 299F, 299I, 320C, 320H, 320K, 320L, 320Q, 320R, 320S, and 320Y. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1082 and one or more residue differences as compared to SEQ ID NO: 1082, selected from L90F, I92A, I92L, I92P, I92V, W122F, W122L, L129F, L129I, H141 Y, D143A, D143K, W144F, W144Y, W145F, W145L, H150D, H150E, H150G, H150R, H150S, H150V, H150W, P151 A, P151 K, P151 Q, P151 R, P151 T/D155T, E152D, E152H, E152L, E152Q, E152R, E152T, E152V, E152Y, A154E, A154K, A154L, A154Q, A154S, A154T, A154V, A154Y, D155E, D155G, D155H, D155Q, D155Q/A265V, D155R, D155S, D155T, K157R, G160I, G160Q, G160V, G160W, K161 C, K161 R, K161 V, D164Q, D164R, D164S, A165G, A165S, F168E, F168K, F168L, F168N, F168R, F168V, V180A, V180C, V180L, V180T, 1181 L, 1181 V, C184A, C184G, C184S, Y193H, Y193L, Y193M, Y193V, K197H, K197R, F204L, F204M, F204V, F204W, F204Y, V205I, V205L, V205L/G472D, V206L, L299F, L299I, A320C, A320H, A320K, A320L, A320Q, A320R, A320S, and A320Y.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1218 and one or more residue differences as compared to SEQ ID NO: 1218, selected from 129, 129/160, 129/160/164, 129/160/184, 129/164, 129/164/184, 129/205, 146/152/244, 150/155/226, 150/155/244, 152, 154, 154/160/184, 154/164, 154/205, 155, 160, 160/164, 160/164/184, 160/184, 164, 164/204, 164/205, 204, 205, 226, 226/244, and 244. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1218 and one or more residue differences as compared to SEQ ID NO: 1218, selected from 1291, 1291/1601, 1291/1601/164Q, 1291/1601/184A, 1291/164Q, 1291/164Q/184A, 1291/2051, 146L/152T/244I, 150D/155T/226R, 150D/155T/244I, 152T, 154K, 154K/1601/184A, 154K/164Q, 154K/205I, 155T, 1601, 1601/164Q, 1601/164Q/184A, 1601/184A, 164Q, 164Q/204L, 164Q/205I, 204L, 2051, 226R, 226R/244I, and 2441. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1218 and one or more residue differences as compared to SEQ ID NO: 1218, selected from L129I, L129I/G160I, L129I/G160I/D164Q, L129I/G160I/C184A, L129I/D164Q, L129I/D164Q/C184A, L129I/V205I, M146L/E152T/V244I, H150D/D155T/E226R, H150D/D155T/V244I, E152T, A154K, A154K/G160I/C184A, A154K/D164Q, A154K/V205I, D155T, G160I, G160I/D164Q, G160I/D164Q/C184A, G160I/C184A, D164Q, D164Q/F204L, D164Q/V205I, F204L, V205I, E226R, E226R/V244I, and V244I. In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1218 and one or more residue differences as compared to SEQ ID NO: 1218, selected from 129, 129/143, 129/154/164, 129/154/164/205, 129/154/205, 129/160, 129/160/164, 129/160/205, 129/164/205, 143, 143/150/155/226/244, 143/150/155/244, 143/150/226, 143/150/226/244/343, 143/150/226/343, 143/150/244, 143/150/343, 143/152, 143/152/226, 143/152/226/244, 143/152/226/343, 143/152/343, 143/155, 143/155/226/244, 143/155/226/244/343, 143/155/343, 143/226, 143/226/244/343, 143/226/343, 143/244, 143/244/343, 143/343, 146/152/244, 150/155/226, 150/155/244, 150/155/244/343, 150/226/244/343, 152, 152/174/244/343, 152/226, 152/226/343, 152/343, 154, 154/160/184, 154/164, 154/164/204, 154/164/204/205, 154/205, 155, 160, 160/164, 160/164/184, 164, 205, 226, 226/244, and 244. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1218 and one or more residue differences as compared to SEQ ID NO: 1218, selected from 1291, 1291/143H, 1291/154K/164Q, 1291/154K/164Q/205I, 1291/154K/205I, 1291/1601, 1291/1601/164Q, 1291/1601/2051, 1291/164Q/2051, 143R, 143R/150D/155T/226R/244I, 143R/150D/155T/244I, 143R/150D/226R, 143R/150D/226R/244I/343R, 143R/150D/226R/343R, 143R/150D/244I, 143R/150D/343R, 143R/152T, 143R/152T/226R, 143R/152T/226R/244I, 143R/152T/226R/343R, 143R/152T/343R, 143R/155T, 143R/155T/226R/244I, 143R/155T/226R/244I/343R, 143R/155T/343R, 143R/226R, 143R/226R/244I/343R, 143R/226R/343R, 143R/244I, 143R/244I/343R, 143R/343R, 146L/152T/2441 , 150D/155T/226R, 150D/155T/244I, 150D/155T/244I/343R, 150D/226R/244I/343R, 152T, 152T/174R/244I/343R, 152T/226R, 152T/226R/343R, 152T/343R, 154K, 154K/1601/184A, 154K/164Q, 154K/164Q/204L, 154K/164Q/204L/205I, 154K/205I, 155T, 1601, 1601/164Q, 1601/164Q/184A, 164Q, 205I, 226R, 226R/244I, and 244I. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1218 and one or more residue differences as compared to SEQ ID NO: 1218, selected from L129I, L129I/D143H, L129I/A154K/D164Q, L129I/A154K/D164Q/V205I, L129I/A154K/V205I , L129I/G160I, L129I/G160I/D164Q, L129I/G160I/V205I, L129I/D164Q/V205I, D143R, D143R/H150D/D155T/E226R/V244I, D143R/H150D/D155T/V244I, D143R/H150D/E226R, D143R/H150D/E226R/V244I/E343R, D143R/H150D/E226R/E343R, D143R/H150D/V244I, D143R/H150D/E343R, D143R/E152T, D143R/E152T/E226R, D143R/E152T/E226R/V244I, D143R/E152T/E226R/E343R, D143R/E152T/E343R, D143R/D155T, D143R/D155T/E226R/V244I, D143R/D155T/E226R/V244I/E343R, D143R/D155T/E343R, D143R/E226R, D143R/E226R/V244I/E343R, D143R/E226R/E343R, D143R/V244I, D143R/V244I/E343R, D143R/E343R, M146L/E152T/V244I, H150D/D155T/E226R, H150D/D155T/V244I, H150D/D155T/V244I/E343R, H150D/E226R/V244I/E343R, E152T, E152T/G174R/V244I/E343R, E152T/E226R, E152T/E226R/E343R, E152T/E343R, A154K, A154K/G160I/C184A, A154K/D164Q, A154K/D164Q/F204L, A154K/D164Q/F204L/V205I, A154K/V205I, D155T, G160I, G160I/D164Q, G160I/D164Q/C184A, D164Q, V205I, E226R, E226R/V244I, and V244I.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 98/129/157/168, 129/157/168, 151 , 151/152/255, 151/152/273, 151 /255, 151 /343, 157, 157/164, 157/164/168, 157/168, 164, 164/255, 168, 183, 255, 255/343, and 343. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 981/1291/157R/168K, 1291/157R/168K, 151 Q, 151 Q/152T/255R, 151 Q/152T/273Y, 151 Q/255R, 151 R/343R, 157R, 157R/164D, 157R/164R, 157R/164R/168K, 157R/168K, 164D, 164D/255R, 168K, 183I, 255R, 255R/343R, and 343R. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from L98I/L129I/K157R/Q168K, L129I/K157R/Q168K, P151 Q, P151 Q/E152T/A255R, P151 Q/E152T/D273Y, P151 Q/A255R, P151 R/E343R, K157R, K157R/Q164D, K157R/Q164R, K157R/Q164R/Q168K, K157R/Q168K, Q164D, Q164D/A255R, Q168K, V183I, A255R, A255R/E343R, and E343R.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 129, 129/157, 129/157/168, 129/164, 129/164/168/183, 129/164/183, 143, 143/151 , 143/151/152, 143/151/164, 143/164, 143/164/255, 143/164/343, 143/255, 143/343, 151 , 151/152, 151/152/164/343, 151/152/343, 151 /255, 151/255/343, 151/343, 152, 152/255, 157, 157/164/168, 157/168, 164, 164/168, 164/255, 164/447, 168, 183, 255, 255/343, 298/343, and 343. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 1291, 1291/157R, 1291/157R/168K, 1291/164R, 1291/164R/168K/183M, 1291/164R/183I, 143R, 143R/151 Q, 143R/151 R, 143R/151 R/152T, 143R/151 R/164D, 143R/164D, 143R/164D/255R, 143 R/164 D/343 R, 143R/255R, 143R/343R, 151 R, 151 R/152T, 151 R/152T/164D/343R, 151 R/152T/343R, 151 R/255R, 151 R/255R/343R, 151 R/343R, 152T, 152T/255R, 157R, 157R/164R/168K, 157R/168K, 164D, 164D/255R, 164R, 164R/168K, 164R/447T, 168K, 1831, 255R, 255R/343R, 298I/343R, and 343R. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from L129I, L129I/K157R, L129I/K157R/Q168K, L129I/Q164R, L129I/Q164R/Q168K/V183M, L129I/Q164R/V183I, D143R, D143R/P151 Q, D143R/P151 R, D143R/P151 R/E152T,
D143R/P151 R/Q164D, D143R/Q164D, D143R/Q164D/A255R, D143R/Q164D/E343R, D143R/A255R, D143R/E343R, P151 R, P151 R/E152T, P151 R/E152T/Q164D/E343R, P151 R/E152T/E343R, P151 R/A255R, P151 R/A255R/E343R, P151 R/E343R, E152T, E152T/A255R, K157R,
K157R/Q164R/Q168K, K157R/Q168K, Q164D, Q164D/A255R, Q164R, Q164R/Q168K, Q164R/A447T, Q168K, V183I, A255R, A255R/E343R, V298I/E343R, and E343R.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 69, 69/164, 72, 73, 91 , 92/164, 95, 96, 97, 98, 1 10, 1 12, 1 13, 121 , 121/164, 122, 122/164, 124, 126, 127, 139, 140, 141 , 144, 144/164, 145, 146, 147, 148, 150, 150/164, 151 , 151/164, 153, 153/164, 155, 156, 157, 159, 160, 161 , 163, 164, 164/172, 164/173, 164/182, 164/188, 164/191 , 164/192, 164/195, 164/197, 164/199, 164/202, 164/205/249, 164/209, 165, 166, 167, 169, 170, 171 , 172, 173, 177, 184, 187, 188, 190, 191 , 192, 193, 194, 195, 197, 197/266, 198, 199, 200, 202, 203, 204, 205, 208, 209, 210, 321 , 324, 331 , and 349. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 69Q, 69V/164D, 72L, 72Q, 73Q, 73W, 91 C, 91 L, 91 V, 92L/164D, 95G, 95S, 95V, 96G, 97T, 98V, 1 1 OR, 1 10T, 1 10V, 1 12A, 1 12H, 1 13V, 121 A, 121 L/164D, 121 Q, 122C, 122F, 122L, 122V, 122V/164D, 124L, 124M, 126A, 126S, 127A, 1271, 1391, 140L, 140V, 141 T, 144F, 144L, 144L/164D, 144V, 145F, 145G, 145K, 145L, 145Q, 145R, 145S, 145T, 146A, 146F, 146G, 146K, 146Q, 146R, 146V, 147W, 148A, 148E, 148F, 148G, 148R, 148V, 150A, 150A/164D, 150G, 150L, 150L/164D, 150R, 150R/164D, 150S/164D, 150T, 150V, 150W, 151 A, 151 C, 151 E, 151 G, 151 1, 151 K, 151 L, 151 L/164D, 151 M, 151 N, 151 Q, 151 S, 151 T, 151 V, 151 W, 151 Y, 151 Y/164D, 153G, 153G/164D, 153N, 153P, 153R, 155F, 156C, 156G, 156L, 156Y, 157A, 157C, 157H, 157P, 157Q, 157R, 157S, 157V, 159A, 159G, 159P, 159Q, 159T, 160A, 160L, 160Q, 160S, 160V, 160Y, 161 A, 161 C, 161 F, 161 L, 161 M, 161 T, 163D, 163G, 163K, 163N, 163Q, 163W, 164D, 164D/172M, 164D/173M, 164D/182W, 164D/188G, 164D/191 R, 164D/192E, 164D/195R, 164D/197A, 164D/199G, 164D/199L, 164D/202A, 164D/205L/249E, 164D/209R, 165H, 165T, 165V, 166M, 166R, 166V, 166W, 167L, 167R, 167V, 1691, 169S, 169T, 169Y, 170L, 171 1, 171 R, 171 S, 1721, 172S, 172T, 172V, 1731, 177G, 177R, 177S, 177T, 177V, 177W, 177Y, 184E, 184G, 184Q, 184S, 184T, 187G, 188G, 190D, 190G, 190M, 190W, 191 G, 191 L, 191 W, 192A, 192L, 193W, 194C, 194R, 194W, 195G, 195H, 195W, 197A, 197L/266V, 197M, 197S, 198A, 198G, 1981, 198K, 198L, 198N, 198R, 199G, 199L, 199W, 199Y, 200A, 200F, 200L, 200R, 200V, 200Y, 202F, 202I, 202L, 202S, 202V, 203Q, 204I, 204T, 204V, 205T, 208C, 209A, 209G, 209V, 2101, 21 OP, 21 OR, 210T, 210V, 321 A, 321 S, 324M, 331 L, 349K, and 349L. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from E69Q, E69V/Q164D, R72L, R72Q, K73Q, K73W, A91 C, A91 L, A91 V, I92L/Q164D, T95G, T95S, T95V, T96G, S97T, L98V, K1 10R, K1 10T, K1 10V, G1 12A, G1 12H, S1 13V, W121 A, W121 L/Q164D, W121 Q, W122C, W122F, W122L, W122V, W122V/Q164D, H124L, H124M, T126A, T126S, L127A, L127I, L139I, I140L, 1140V, H141 T, W144F, W144L, W144L/Q164D, W144V, W145F, W145G, W145K, W145L, W145Q, W145R, W145S, W145T, M146A, M146F, M146G, M146K, M146Q, M146R, M146V, R147W, P148A, P148E, P148F, P148G, P148R, P148V, H150A, H150A/Q164D, H150G, H150L, H150L/Q164D, H150R, H150R/Q164D, H150S/Q164D, H150T, H150V, H150W, P151 A, P151 C, P151 E, P151 G, P151 1, P151 K, P151 L, P151 L/Q164D, P151 M, P151 N, P151 Q, P151 S, P151 T, P151 V, P151 W, P151 Y, P151 Y/Q164D, D153G, D153G/Q164D, D153N, D153P, D153R, D155F, E156C, E156G, E156L, E156Y, K157A, K157C, K157H, K157P, K157Q, K157R, K157S, K157V, S159A, S159G, S159P, S159Q, S159T, G160A, G160L, G160Q, G160S, G160V, G160Y, K161 A, K161 C, K161 F, K161 L, K161 M, K161 T, A163D, A163G, A163K, A163N, A163Q, A163W, Q164D, Q164D/R172M, Q164D/L173M, Q164D/S182W, Q164D/A188G, Q164D/Q191 R, Q164D/S192E, Q164D/Q195R, Q164D/K197A, Q164D/S199G, Q164D/S199L, Q164D/E202A, Q164D/V205L/K249E, Q164D/K209R, A165H, A165T, A165V, S166M, S166R, S166V, S166W, H167L, H167R, H167V, C169I, C169S, C169T, C169Y, R170L, Q171 1, Q171 R, Q171 S, R172I, R172S, R172T, R172V, L173I, M177G, M177R, M177S, M177T, M177V, M177W, M177Y, C184E, C184G, C184Q, C184S, C184T, E187G, A188G, I190D, I190G, I190M, I190W, Q191 G, 0191 L, Q191 W, S192A, S192L, Y193W, L194C, L194R, L194W, Q195G, Q195H, Q195W, K197A, K197L/A266V, K197M, K197S, Q198A, Q198G, Q198I, Q198K, Q198L, Q198N, Q198R, S199G, S199L, S199W, S199Y, H200A, H200F, H200L, H200R, H200V, H200Y, E202F, E202I, E202L, E202S, E202V, R203Q, F204I, F204T, F204V, V205T, A208C, K209A, K209G, K209V, H210I, H210P, H210R, H210T, H210V, R321 A, R321 S, V324M, W331 L, E349K, and E349L.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 69, 69/164, 72, 73, 91 , 92/164, 95, 97, 98, 121/164, 126, 127, 139, 140, 142, 144, 144/164, 145, 146, 146/164, 148, 150, 150/164, 151 , 153, 153/164, 155, 156, 157, 160, 163, 164/173, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/205/249, 164/209, 165, 166, 167, 169, 171 , 172, 173, 177, 182, 187, 188, 190, 191 , 192, 194, 195, 197/266, 198, 199, 200, 202, 203, 204, 205, 208, 209, 210, 324, 331 , and 349. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 69Q, 69V, 69V/164D, 72L, 72Q, 73Q, 73W, 91 C, 91 V, 92L/164D, 95S, 97T, 98M, 98V, 121 L/164D, 126A, 126S, 1271, 1391, 140L, 140V, 142C, 144F, 144L/164D, 144V, 144Y, 145F, 145K, 145L, 146A, 146F, 146K, 146L/164D, 146Q, 146R, 146V, 148R, 150A, 150A/164D, 150G, 150L, 150L/164D, 150R, 150R/164D, 150S/164D, 150T, 150V, 150W, 151 C, 151 E, 151 K, 151 M, 151 N, 151 Q, 151 S, 151 V, 151 W, 151 Y, 153G, 153G/164D, 153N, 153R, 155C, 155F, 155L, 155S, 155T, 156C, 156G, 156L, 156R, 156S, 156T, 156Y, 157A, 157C, 157P, 157Q, 157S, 157V, 160A, 160L, 160Q, 160S, 160V, 160Y, 163D, 163G, 163K, 163N, 163Q, 163W, 164D/173M, 164D/188G, 164D/191 R, 164D/192E, 164D/195R, 164D/199G, 164D/199L, 164D/205L/249E, 164D/209R, 165G, 165T, 166M, 166V, 167L, 167R, 167V, 1691, 169S, 169T, 1711, 171 R, 171 S, 171 V, 1721, 172T, 172V, 1731, 177G, 177R, 177S, 177T, 177V, 177W, 177Y, 182L, 187G, 188G, 190M, 191 F, 191 G, 192A, 192L, 194W, 195G, 195H, 195M, 195W, 195Y, 197L/266V, 198A, 198L, 198N, 198R, 199F, 199G, 199L, 199W, 199Y, 200A, 200F, 200L, 200R, 200S, 200V, 200Y, 202L, 203Q, 204I, 204T, 204V, 205T, 208S, 209V, 210T, 210V, 324M, 331 L, and 349K. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from E69Q, E69V, E69V/Q164D, R72L, R72Q, K73Q, K73W, A91 C, A91 V, I92L/Q164D, T95S, S97T, L98M, L98V, W121 L/Q164D, T126A, T126S, L127I, L139I, I140L, 1140V, Q142C, W144F, W144L/Q164D, W144V, W144Y, W145F, W145K, W145L, M146A, M146F, M146K, M146L/Q164D, M146Q, M146R, M146V, P148R, H150A, H150A/Q164D, H150G, H150L, H150L/Q164D, H150R, H150R/Q164D, H150S/Q164D, H150T, H150V, H150W, P151 C, P151 E, P151 K, P151 M, P151 N, P151 Q, P151 S, P151 V, P151W, P151Y, D153G, D153G/Q164D, D153N, D153R, D155C, D155F, D155L, D155S, D155T, E156C, E156G, E156L, E156R, E156S, E156T, E156Y, K157A, K157C, K157P, K157Q, K157S, K157V, G160A, G160L, G160Q, G160S, G160V, G160Y, A163D, A163G, A163K, A163N, A163Q, A163W, Q164D/L173M, Q164D/A188G, Q164D/Q191 R, Q164D/S192E, Q164D/Q195R, Q164D/S199G, Q164D/S199L, Q164D/V205L/K249E, Q164D/K209R, A165G, A165T, S166M, S166V, H167L, H167R, H167V, C169I, C169S, C169T, Q1711, Q171 R, Q171 S, Q171 V, R172I, R172T, R172V, L173I, M177G, M177R, M177S, M177T, M177V, M177W, M177Y, S182L, E187G, A188G, I190M, Q191 F, Q191 G, S192A, S192L, L194W, Q195G, Q195H, Q195M, Q195W, Q195Y, K197L/A266V, Q198A, Q198L, Q198N, Q198R, S199F, S199G, S199L, S199W, S199Y, H200A, H200F, H200L, H200R, H200S, H200V, H200Y, E202L, R203Q, F204I, F204T, F204V, V205T, A208S, K209V, H210T, H210V, V324M, W331 L, and E349K.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 73, 91 , 93, 95, 97, 98, 110, 112, 122, 122/164, 124, 127, 139, 144, 145, 146, 146/164, 147, 148, 150, 150/164, 151 , 151/164, 153, 153/164, 155, 156, 157, 159, 160, 161 , 162, 163, 164, 164/182, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/205/249, 164/209, 165, 166, 167, 169, 171 , 172, 177, 184, 187, 188, 190, 191 , 192, 193, 195, 197/266, 198, 199, 200, 204, 205, 206, 207, 208, 209, 210, and 324. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 73Q, 73W, 91 C, 91 L, 93S, 95G, 95R, 95S, 95V, 97T, 98V, 110T, 110V, 112A, 112H, 122C, 122F, 122L, 122V, 122V/164D, 124A, 124L, 124M, 1271, 1391, 144F, 144Y, 145F, 145L, 145Q, 145R, 145S, 145T, 146A, 146F, 146G, 146K, 146L/164D, 146Q, 146R, 146V, 147W, 148E, 148F, 148G, 148R, 148V, 150A, 150G, 150G/164D, 150L, 150R/164D, 150S/164D, 150T, 150V, 150W, 151 A, 151 C, 151 E, 151 G, 151 1, 151 K, 151 L, 151 L/164D, 151 M, 151 N, 151 Q, 151 S, 151 T, 151 V, 151 W, 151 Y, 151 Y/164D, 153G, 153G/164D, 153P, 153Q, 153R, 155L, 155S, 155W, 156G, 156S, 156Y, 157A, 157C, 157H, 157P, 157R, 157S, 157V, 159A, 159G, 159P, 159Q, 159T, 160A, 160L, 160M, 160S, 160V, 160Y, 161 A, 161 C, 161 F, 161 L, 161 M, 161 T, 162Y, 163D, 163G, 163W, 164D, 164D/182W, 164D/188G, 164D/191 R, 164D/192E, 164D/195R, 164D/199G, 164D/199L, 164D/205L/249E, 164D/209R, 165G, 165T, 165V, 166M, 166V, 166W, 167L, 1691, 169T, 171 R, 171 S, 171 V, 1721, 177W, 184Q, 184T, 187G, 188G, 190D, 190G, 190M, 190W, 191 F, 191 L, 191 W, 192A, 193W, 195H, 195R, 195Y, 197L/266V, 198A, 198L, 198N, 198R, 198V, 199G, 199L, 199W, 199Y, 200R, 204L, 205T, 206L, 207K, 207Q, 208S, 208V, 209A, 209G, 209V, 21 OP, 21 OR, 21 OT, 210V, and 324M. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from K73Q, K73W, A91 C, A91 L, D93S, T95G, T95R, T95S, T95V, S97T, L98V, K1 10T, K1 10V, G1 12A, G1 12H, W122C, W122F, W122L, W122V, W122V/Q164D, H124A, H124L, H124M, L127I, L139I, W144F, W144Y, W145F, W145L, W145Q, W145R, W145S, W145T, M146A, M146F, M146G, M146K, M146L/Q164D, M146Q, M146R, M146V, R147W, P148E, P148F, P148G, P148R, P148V, H150A, H150G, H150G/Q164D, H150L, H150R/Q164D, H150S/Q164D, H150T, H150V, H150W, P151 A, P151 C, P151 E, P151 G, P151 1, P151 K, P151 L, P151 L/Q164D, P151 M, P151 N, P151 Q, P151 S, P151 T, P151 V, P151 W, P151 Y, P151 Y/Q164D, D153G, D153G/Q164D, D153P, D153Q, D153R, D155L, D155S, D155W, E156G, E156S, E156Y, K157A, K157C, K157H, K157P, K157R, K157S, K157V, S159A, S159G, S159P, S159Q, S159T, G160A, G160L, G160M, G160S, G160V, G160Y, K161 A, K161 C, K161 F, K161 L, K161 M, K161 T, W162Y, A163D, A163G, A163W, Q164D, Q164D/S182W, Q164D/A188G, Q164D/Q191 R, Q164D/S192E, Q164D/Q195R, Q164D/S199G, Q164D/S199L, Q164D/V205L/K249E, Q164D/K209R, A165G, A165T, A165V, S166M, S166V, S166W, H167L, C169I, C169T, Q171 R, Q171 S, Q171 V, R172I, M177W, C184Q, C184T, E187G, A188G, I190D, I190G, I190M, I190W, Q191 F, 0191 L, Q191 W, S192A, Y193W, Q195H, Q195R, Q195Y, K197L/A266V, Q198A, Q198L, Q198N, Q198R, Q198V, S199G, S199L, S199W, S199Y, H200R, F204L, V205T, V206L, R207K, R207Q, A208S, A208V, K209A, K209G, K209V, H210P, H210R, H210T, H210V, and V324M.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 69, 73, 91 , 127, 140, 144, 145, 146, 146/164, 150, 150/164, 151 , 153, 155, 156, 157, 160, 163, 164, 164/172, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/209, 165, 167, 169, 171 , 172, 182, 183, 188, 191 , 192, 193, 195, 196, 198, 199, 200, 204, 205, 208, 210, and 324. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from 69G, 69V, 73Q, 73W, 91 G, 1271, 140V, 144F, 144Y, 145R, 146A, 146F, 146K, 146L/164D, 146Q, 146R, 150G, 150L, 150R, 150R/164D, 150S/164D, 150W, 151 K, 151 N, 153G, 153Q, 153R, 155C, 155F, 155L, 155M, 155P, 155S, 155T, 155W, 156C, 156G, 156R, 156S, 156T, 156W, 156Y, 157A, 157P, 157R, 157S, 157V, 160A, 160L, 160M, 160Q, 160R, 160S, 160V, 160Y, 163D, 163K, 163N, 163W, 164D, 164D/172M, 164D/188G, 164D/191 R, 164D/192E, 164D/195R, 164D/199G, 164D/199L, 164D/209R, 165G, 167L, 167R, 169A, 1691, 169T, 171 H, 171 R, 171 S, 171 V, 1721, 182T, 183L, 188G, 191 F, 191 G, 191 W, 192A, 192L, 192R, 193W, 195G, 195H, 195M, 195R, 195W, 195Y, 196C, 196G, 196L, 196W, 198A, 198L, 198N, 198R, 198V, 199L, 199W, 199Y, 200R, 200Y, 204L, 205T, 208S, 210V, and 324M. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 1580 and one or more residue differences as compared to SEQ ID NO: 1580, selected from E69G, E69V, K73Q, K73W, A91 G, L127I, 1140V, W144F, W144Y, W145R, M146A, M146F, M146K, M146L/Q164D, M146Q, M146R, H150G, H150L, H150R, H150R/Q164D, H150S/Q164D, H150W, P151 K, P151 N, D153G, D153Q, D153R, D155C, D155F, D155L, D155M, D155P, D155S, D155T, D155W, E156C, E156G, E156R, E156S, E156T, E156W, E156Y, K157A, K157P, K157R, K157S, K157V, G160A, G160L, G160M, G160Q, G160R, G160S, G160V, G160Y, A163D, A163K, A163N, A163W, Q164D, Q164D/R172M, Q164D/A188G, Q164D/Q191 R, Q164D/S192E, Q164D/Q195R, Q164D/S199G, Q164D/S199L, Q164D/K209R, A165G, H167L, H167R, C169A, C169I, C169T, Q171 H, Q171 R, Q171 S, Q171 V, R172I, S182T, V183L, A188G, Q191 F, Q191 G, Q191 W, S192A, S192L, S192R, Y193W, Q195G, Q195H, Q195M, Q195R, Q195W, Q195Y, D196C, D196G, D196L, D196W, Q198A, Q198L, Q198N, Q198R, Q198V, S199L, S199W, S199Y, H200R, H200Y, F204L, V205T, A208S, H210V, and V324M.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from 38/143/168/220/290/317/423, 38/152/154/164/168/220/290/317/423, 38/152/168/220/290/317/343/423, 38/152/168/220/290/317/423, 38/154/164/168/220/290/317/423, 38/168/220/290/317/423, and 38/220/317. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from 38F/143R/168Q/220R/290R/317M/423D, 38F/152T/154K/164R/168K/220R/290R/317M/423D, 38F/152T/168Q/220R/290R/317M/343R/423D, 38F/152T/168Q/220R/290R/317M/423D, 38F/154K/164Q/168Q/220R/290R/317M/423D, 38F/168Q/220R/290R/317M/423D, and 38F/220R/317M. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from C38F/D143R/F168Q/P220R/K290R/R317M/W423D,
C38F/E152T/A154K/D164R/F168K/P220R/K290R/R317M/W423D,
C38F/E152T/F168Q/P220R/K290R/R317M/E343R/W423D, C38F/E152T/F168Q/P220R/K290R/R317M/W423D,
C38F/A154K/D164Q/F168Q/P220R/K290R/R317M/W423D, C38F/F168Q/P220R/K290R/R317M/W423D, and C38F/P220R/R317M.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2368 and one or more residue differences as compared to SEQ ID NO: 2368, selected from 73/143/169/191/193, 73/156, 73/156/192/435, 73/156/210, 73/163, 73/163/210, 73/192/193, 127, 127/160/172/198, 127/172, 127/198, 146, 146/151/188, 146/324, 151/171/324, 151/324, 156/169, 156/192, 157/171 , 163, 163/169, 163/169/191/192/193, 165/171/324, 165/188/324, 165/324, 169, 171/188/324, 171/324, 172, 188, 192, 192/193, 198, 210, and 324. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2368 and one or more residue differences as compared to SEQ ID NO: 2368, selected from 73Q/143K/1691/191 F/193W, 73Q/156R, 73Q/156R/210V, 73Q/156Y/192A/435K, 73Q/163D, 73Q/163D/210Q, 73Q/163N, 73Q/163N/210V, 73Q/192A/193W, 1271, 1271/160A/1721/198R, 1271/1721, 1271/198R, 146L, 146L/151 N/188G , 146L/324M, 151 N/171 V/324M, 151 N/324M, 156Y/169I, 156Y/192E, 157P/171 V, 163D, 163D/1691/191 F/192A/193W, 163N/169I, 165G/171 R/324M, 165G/188G/324M, 165G/324M, 1691, 171 R/324M, 171 V/188G/324M, 171 V/324M, 1721, 188G, 192A, 192E/193W, 198R, 210V, and 324M. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2368 and one or more residue differences as compared to SEQ ID NO: 2368, selected from K73Q/D143K/C169I/Q191 F/Y193W, K73Q/E156R, K73Q/E156R/H210V, K73Q/E156Y/S192A/M435K, K73Q/A163D, K73Q/A163D/H210Q, K73Q/A163N, K73Q/A163N/H210V, K73Q/S192A/Y193W, L127I, L127I/G160A/R172I/Q198R, L127I/R172I, L127I/Q198R, M146L, M146L/P151 N/A188G, M146L/V324M, P151 N/Q171 V/V324M, P151 N/V324M, E156Y/C169I, E156Y/S192E, K157P/Q171 V, A163D, A163D/C169I/Q191 F/S192A/Y193W, A163N/C169I, A165G/Q171 R/V324M, A165G/A188G/V324M, A165G/V324M, C169I, Q171 R/V324M, Q171 V/A188G/V324M, Q171 V/V324M, R172I, A188G, S192A, S192E/Y193W, Q198R, H210V, and V324M.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2368 and one or more residue differences as compared to SEQ ID NO: 2368, selected from 73, 73/163/191/192/193, 127/160/172, 127/160/172/198, 144, 160, 160/172, 163/191/192, and 192. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2368 and one or more residue differences as compared to SEQ ID NO: 2368, selected from 73Q,
73Q/163N/191 F/192A/193W, 1271/160A/172I, 1271/160A/1721/198R, 144Y, 160A, 160A/172I, 163N/191 F/192A, and 192A. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2368 and one or more residue differences as compared to SEQ ID NO: 2368, selected from K73Q, K73Q/A163N/Q191 F/S192A/Y193W, L127I/G160A/R172I, L127I/G160A/R172I/Q198R, W144Y, G160A, G160A/R172I, A163N/Q191 F/S192A, and S192A.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2426 and one or more residue differences as compared to SEQ ID NO: 2426, selected from 8, 13, 17, 18, 20/146/157/167, 26, 34/38, 38/39/45, 67, 70, 73, 73/150/155/169, 73/150/171/192, 73/155, 73/155/169/199, 73/155/171 , 73/192, 73/199, 78, 82, 84, 86, 86/384, 89, 90, 91 , 95, 102, 110, 111 , 113, 114, 115, 122/127, 126/127, 127/131/165, 127/133, 143, 143/146, 143/146/153, 143/146/157, 143/146/167, 143/146/167/188, 143/146/188, 143/153/167/188/246, 143/156/157/167/195, 143/156/188, 143/188, 143/188/195, 143/195, 144, 145, 145/151 , 146, 146/152, 146/153/157, 146/153/157/167, 146/153/167, 146/157, 146/157/167, 146/167, 150, 150/155/169, 150/155/169/192, 150/155/192, 150/169, 150/192, 150/199, 151/152/154, 152/154, 152/154/155/160, 153/157/167, 155/171 , 155/192, 156/270, 160/371 , 167/188, 168/172, 169, 184, 190, 192/198, 198, 206, 208, 209, 210, 214/220, 215/220, 216/220, 217/220, 219/220, 220, 220/222, 220/225, 220/226, 231 , 235, 242, 257, 259, 261 , 262, 267, 268, 269, 270, 272, 274, 275, 276, 277, 278, 282, 283/290, 284/290, 287/290, 288/290, 289/290, 290, 290/291 , 290/292, 290/293, 290/294, 304, 305, 305/373, 306, 307, 309, 317, 329, 333, 334, 339, 341 , 344, 345, 346, 352, 353, 355, 358, 366, 368, 370, 372, 373, 375, 376, 378, 379, 380, 381 , 382, 383, 384, 385, 386/393, 387/393, 389/393, 390/393, 391/393, 392/393, 393/394, 414, 415, 420/423, 422/423, 423, 423/425, 432, 440, 441 , 443, 469, and 472. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2426 and one or more residue differences as compared to SEQ ID NO: 2426, selected from 8D, 8V, 13W, 17V, 18D, 18L, 18P, 20D/146C/157P/167L, 26R, 34H/38C, 38C/39F/45I, 38C/39P/45I, 67G, 70G, 73H, 73Q/150N/155L/169T, 73Q/150N/171 R/192A, 73Q/155L, 73Q/155L/169T/199L, 73Q/155L/171 R, 73Q/192A, 73Q/199L, 73R, 78W, 82Q, 82R, 84T, 86F/384T, 86I, 89M, 90W, 91 L, 91 V, 95V, 102G, 110L, 111 M, 113A, 113G, 113V, 114D, 114T, 115Q, 122A/127L, 126V/127L, 127L/131 A/165T, 127L/133M, 143T, 143V, 143V/146C, 143V/146C/153G, 143V/146C/157P, 143V/146C/167L, 143V/146C/167L/188G, 143V/146C/188G, 143V/146K, 143V/153G/167L/188G/246R, 143V/156R/157P/167L/195H, 143V/156R/188G, 143V/188G, 143V/188G/195H, 143V/195H, 144F, 145L, 145V/151 R, 146A/152A, 146C, 146C/153G/157P, 146C/153G/157P/167L, 146C/153G/167L, 146C/157P, 146C/157P/167L, 146C/167L, 146K, 146K/157P, 150N, 150N/155L/169T, 150N/155L/169T/192A, 150N/155L/192A, 150N/169T, 150N/192A, 150N/199L, 151 E/152E/154A, 151 R/152E/154A, 151T/152E/154A, 152E/154A, 152E/154A/155N/160G, 153G/157P/167L, 155L/171 R, 155L/192A, 156G/270P, 160G/371 W, 167L/188G, 168F/172V, 169T, 184A, 190G, 190S, 192L/198Q, 198D, 198G, 206F, 206W, 208V, 209G, 209M, 210G, 21 OL, 214G/220P, 214V/220P, 215F/220P, 216I/220P, 217D/220P, 217G/220P, 217L/220P, 217R/220P, 219G/220P, 220L, 220P/222C, 220P/222H, 220P/222R, 220P/225M, 220P/226R, 220P/226S, 231 G, 235S, 242S, 257G, 259H, 261 K, 262L, 267L, 268W, 269A, 269E, 269G, 270D, 270L, 270P, 272G, 272R, 272T, 274K, 274R, 275R, 275W, 276A, 276E, 276G, 277V, 278V, 282M, 283S/290K, 284A/290K, 284S/290K, 287P/290K, 288M/290K, 288P/290K, 288R/290K, 288V/290K, 289S/290K, 290C, 290K, 290K/291 E, 290K/291G, 290K/291 S, 290K/292A, 290K/293L, 290K/294K, 290K/294M, 290K/294R, 290K/294S, 290K/294W, 290Q, 290V, 304E, 304V, 305G/373C, 305R, 306G, 307S, 309V, 317R, 329A, 329R, 329T, 333L, 334L, 339G, 341 L, 344G, 345T, 346T, 346V, 352L, 353V, 355C, 358C, 366S, 368W, 370S, 372G, 373T, 375Y, 376L, 378A, 378G, 378P, 378S, 379P, 380T, 380V, 381 S, 382L, 383F, 383R, 384N, 384V, 385L, 386A/393E, 386S/393E, 386T/393E, 386V/393E, 387A/393E, 387R/393E, 389L/393E, 389M/393E, 389V/393E, 390E/393E, 390G/393E, 391 L/393E, 392P/393E, 393E/394R, 414G, 415P, 415V, 420E/423W, 422G/423W, 422S/423W, 422V/423W, 423A, 423W/425A, 432T, 440G, 441 Q, 441 S, 441 W, 443P, 469A, 472A, and 472R. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2426 and one or more residue differences as compared to SEQ ID NO: 2426, selected from Q8D, Q8V, H13W, A17V, A18D, A18L, A18P, G20D/M146C/K157P/H167L, K26R, D34H/F38C, F38C/S39F/V45I, F38C/S39P/V45I, S67G, V70G, K73H, K73Q/H150N/D155L/C169T, K73Q/H150N/Q171 R/S192A, K73Q/D155L, K73Q/D155L/C169T/S199L, K73Q/D155L/Q171 R, K73Q/S192A, K73Q/S199L, K73R, H78W, L82Q, L82R, Q84T, Y86F/A384T, Y86I, I89M, L90W, A91 L, A91 V, T95V, H102G, K110L, L111 M, S113A, S113G, S113V, V114D, V114T, K115Q, W122A/I127L, T126V/I127L, I127L/G131 A/A165T, I127L/T133M, D143T, D143V, D143V/M146C, D143V/M146C/D153G, D143V/M146C/K157P, D143V/M146C/H167L, D143V/M146C/H167L/A188G, D143V/M146C/A188G, D143V/M146K, D143V/D153G/H167L/A188G/S246R, D143V/E156R/K157P/H167UQ195H, D143V/E156R/A188G, D143V/A188G, D143V/A188G/Q195H, D143V/Q195H, W144F, W145L, W145V/P151 R, M146A/T152A, M146C, M146C/D153G/K157P, M146C/D153G/K157P/H167L, M146C/D153G/H167L, M146C/K157P, M146C/K157P/H167L, M146C/H167L, M146K, M146K/K157P, H150N, H150N/D155L/C169T, H150N/D155L/C169T/S192A, H150N/D155L/S192A, H150N/C169T, H150N/S192A, H150N/S199L, P151 E/T152E/K154A, P151 R/T152E/K154A, P151 T/T152E/K154A, T152E/K154A, T152E/K154A/D155N/A160G, D153G/K157P/H167L, D155L/Q171 R, D155L/S192A, E156G/K270P, A160G/P371W, H167L/A188G, K168F/I172V, C169T, C184A, I190G, 1190S, S192L/R198Q, R198D, R198G, V206F, V206W, A208V, K209G, K209M, H210G, H210L, I214G/R220P, I214V/R220P, L215F/R220P, D216I/R220P, S217D/R220P, S217G/R220P, S217L/R220P, S217R/R220P, L219G/R220P, R220L, R220P/F222C, R220P/F222H, R220P/F222R, R220P/L225M, R220P/E226R, R220P/E226S, L231 G, T235S, G242S, K257G, S259H, S261 K, V262L, V267L, T268W, L269A, L269E, L269G, K270D, K270L, K270P, K272G, K272R, K272T, E274K, E274R, M275R, M275W, Q276A, Q276E, Q276G, P277V, I278V, Y282M, A283S/R290K, E284A/R290K, E284S/R290K, N287P/R290K, S288M/R290K, S288P/R290K, S288R/R290K, S288V/R290K, P289S/R290K, R290C, R290K, R290K/D291 E, R290K/D291 G, R290K/D291 S, R290K/M292A, R290K/E293L, R290K/P294K, R290K/P294M, R290K/P294R, R290K/P294S, R290K/P294W, R290Q, R290V, P304E, P304V, I305G/Y373C, I305R, E306G, T307S, S309V, M317R, K329A, K329R, K329T, T333L, G334L, R339G, R341 L, E344G, P345T, R346T, R346V, V352L, S353V, L355C, I358C, R366S, A368W, T370S, P372G, Y373T, L375Y, R376L, K378A, K378G, K378P, K378S, G379P, L380T, L380V, V381 S, D382L, A383F, A383R, A384N, A384V, N385L, E386A/A393E, E386S/A393E, E386T/A393E, E386V/A393E, L387A/A393E, L387R/A393E, S389L/A393E, S389M/A393E, S389V/A393E, Q390E/A393E, Q390G/A393E, S391 L/A393E, C392P/A393E, A393E/T394R, N414G, G415P, G415V, S420E/D423W, K422G/D423W, K422S/D423W, K422V/D423W, D423A, D423W/Y425A, G432T, T440G, G441 Q, G441 S, G441 W, A443P, L469A, G472A, and G472R.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2426 and one or more residue differences as compared to SEQ ID NO: 2426, selected from 8, 18, 25, 29, 30, 67, 73, 73/150/155/169, 73/150/155/171 , 73/150/171 /192, 73/171 , 73/192, 73/199, 74, 82, 86, 86/384, 113, 115, 118, 127/128, 143, 150, 150/155/169, 150/155/169/192, 150/155/171 , 150/155/171/199, 150/155/192, 150/155/199, 150/192, 150/199, 155/192, 168/171/172, 195/198, 197/198, 198/201 , 198/203, 198/205, 235, 252, 256, 257, 262, 263, 267/343, 268, 269, 270, 272, 273, 276, 277, 288/290, 289/290, 290, 290/291 , 290/292, 290/294, 298, 304, 305, 307, 333, 346, 355, 366, 370, 386/393, 389/393, 393/395, 440, and 472. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2426 and one or more residue differences as compared to SEQ ID NO: 2426, selected from 8D, 18L, 25G, 29F, 30R, 67G, 73H, 73Q/150N/155L/169T, 73Q/150N/155L/171 R, 73Q/150N/171 R/192A, 73Q/171 R, 73Q/192A, 73Q/199L, 74R, 82F, 82Q, 86A, 86F/384T, 86I, 113E, 115A, 115Q, 118S, 127L/128I, 143H, 150N, 150N/155L/169T, 150N/155L/169T/192A, 150N/155L/171 R, 150N/155L/171 R/199L, 150N/155L/192A, 150N/155L/199L, 150N/192A, 150N/199L, 155L/192A, 168F/171 P/172R, 195K/198Q, 195S/198Q, 197S/198Q, 198Q/201 R, 198Q/203K, 198Q/205T, 235S, 252A, 256E, 256G, 257G, 262L, 263Q, 267P/343G, 268W, 269I, 270P, 272R, 273V, 276E, 277V, 288V/290K, 289S/290K, 290K/291 E, 290K/292A, 290K/294S, 290Q, 290V, 298R, 304W, 305A, 305R, 307S, 333L, 346T, 355K, 366S, 370S, 386S/393E, 389M/393E, 393E/395R, 393E/395W, 440G, and 472R. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2426 and one or more residue differences as compared to SEQ ID NO: 2426, selected from Q8D, A18L, T25G, I29F, E30R, S67G, K73H, K73Q/H150N/D155L/C169T, K73Q/H150N/D155L/Q171 R, K73Q/H150N/Q171 R/S192A, K73Q/Q171 R, K73Q/S192A, K73Q/S199L, A74R, L82F, L82Q, Y86A, Y86F/A384T, Y86I, S113E, K115A, K115Q, A118S, I127L/L128I, D143H, H150N, H150N/D155L/C169T, H150N/D155L/C169T/S192A, H150N/D155L/Q171 R, H150N/D155L/Q171 R/S199L, H150N/D155L/S192A, H150N/D155L/S199L, H150N/S192A, H150N/S199L, D155L/S192A,
K168F/Q171 P/1172R, Q195K/R198Q, Q195S/R198Q, K197S/R198Q, R198Q/N201 R, R198Q/R203K, R198Q/V205T, T235S, N252A, R256E, R256G, K257G, V262L, K263Q, V267P/E343G, T268W, L269I, K270P, K272R, D273V, Q276E, P277V, S288V/R290K, P289S/R290K, R290K/D291 E, R290K/M292A, R290K/P294S, R290Q, R290V, V298R, P304W, I305A, I305R, T307S, T333L, R346T, L355K, R366S, T370S, E386S/A393E, S389M/A393E, A393E/V395R, A393E/V395W, T440G, and G472R.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from 66, 67, 70, 101 , 102, 103, 104, 106, 107, 108, 113, 114, 115, 117, 118, 143, 152, 154, 235, 236/251 , 237, 238, 242, 243, 246, 247, 249, 250, 251 , 256/352, 257, 259, 285, 286, 287, 288, 289, 290, 292, 293, 294, 295, 296, 322, 323, 326, 329, 330, 333, 339, 340, 343, 344, 345, and 352. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from 66T, 67V, 70A, 101 S, 101 T, 102R, 102S, 103V, 104T, 106A, 107P, 107S, 107V, 108M, 113G, 113R, 114A, 1141, 114P, 114S, 115L, 115R, 117A, 118K, 143E, 143G, 143R, 143V, 152R, 154E, 154F, 154Q, 235R, 236V/251 T, 237S, 238L, 242T, 243F, 243L, 246M, 247F, 249F, 249L, 249R, 249V, 249W, 250N, 251 G, 251 R, 251 S, 256K/352A, 257L, 257R, 257V, 259H, 259P, 259Y, 285D, 286G, 286R, 287A, 287G, 287L, 287R, 287V, 288L, 288W, 289I, 289K, 289R, 289S, 290G, 290S, 292L, 292P, 292V, 293G, 294E, 294V, 295F, 295T, 295V, 296A, 296Q, 296R, 322G, 322Q, 322T, 323S, 323T, 326L, 326M, 329G, 329H, 330L, 330V, 333A, 333M, 333R, 333S, 339A, 339G, 339S, 340S, 343P, 343T, 344G, 344M, 345A, 345E, 345K, 345V, 352I, and 352L. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from V66T, S67V, V70A, K101 S, K101T, H102R, H102S, Q103V, V104T, S106A, D107P, D107S, D107V, L108M, S113G, S113R, V114A, V114I, V114P, V114S, K115L, K115R, K117A, A118K, D143E, D143G, D143R, D143V, T152R, K154E, K154F, K154Q, T235R, I236V/K251T, D237S, I238L, G242T, V243F, V243L, S246M, K247F, K249F, K249L, K249R, K249V, K249W, R250N, K251 G, K251 R, K251 S, R256K/V352A, K257L, K257R, K257V, S259H, S259P, S259Y, E285D, T286G, T286R, N287A, N287G, N287L, N287R, N287V, S288L, S288W, P289I, P289K, P289R, P289S, R290G, R290S, M292L, M292P, M292V, E293G, P294E, P294V, L295F, L295T, L295V, K296A, K296Q, K296R, W322G, W322Q, W322T, R323S, R323T, E326L, E326M, K329G, K329H, A330L, A330V, T333A, T333M, T333R, T333S, R339A, R339G, R339S, L340S, E343P, E343T, E344G, E344M, P345A, P345E, P345K, P345V, V352I, and V352L.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from 18, 18/113/270, 18/113/270/329/370, 18/113/270/370/384, 18/113/270/370/412, 18/113/329/384, 18/259/370/384, 18/270, 18/270/370, 18/270/370/384, 18/370, 18/370/384, 73, 73/115, 73/115/184/333, 73/115/184/341 , 73/115/256, 73/184/256, 73/184/273/333, 73/184/333, 73/256, 73/256/375, 73/273, 73/273/333/375, 73/273/375, 73/333, 73/333/375, 73/375, 73/379, 77/233/238/376, 77/233/334/376, 77/262/376, 77/276, 77/334/376, 82, 82/127, 82/127/144/235/290, 82/127/144/266, 82/127/144/266/290, 82/127/144/378, 82/127/235, 82/127/290, 82/144/266/290/378, 82/144/266/378, 82/235/266, 82/235/266/378/383, 82/235/290, 82/235/378, 82/266, 82/266/378, 82/378, 113/270/370/384, 115/256/333/375, 115/256/375, 115/273/375, 115/375, 127/144/290, 127/149/150, 127/266/290/378, 127/378/383, 144/235/290, 144/266/290, 233/334, 233/376, 235, 235/290, 256/333, 262, 266/378, 270/370, 273, 276/352, 290, 290/378, 333, 358, 370, 375, and 378. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from 18D, 18D/113A/270L, 18D/113A/270L/329A/370S, 18D/259R/370S/384V, 18D/270L, 18D/270L/370S, 18D/270U370S/384V, 18D/370S, 18L, 18L/113A/270L, 18L/113A/270L/370S/384V, 18L/113A/329A/384V, 18L/113E/270L/370S/412S, 18L/370S, 18L/370S/384V, 73H, 73H/115Q/184A/341 G, 73H/184A/256G, 73H/273V/333L/375S, 73H/333L, 73R, 73R/115Q, 73R/115Q/184A/333L, 73R/115Q/256G, 73R/184A/273V/333L, 73R/184A/333L, 73R/256G, 73R/256G/375S, 73R/273V, 73R/273V/333L/375S, 73R/273V/375S, 73R/333L, 73R/333L/375S, 73R/375S, 73R/379V, 77V/233U238V/376L, 77V/233L/334L/376L, 77V/262L/376L, 77V/276E, 77V/334L/376L, 82Q, 82Q/127L, 82Q/127L/144Y/235S/290V, 82Q/127L/144Y/266V, 82Q/127L/144 Y/266 V/290 V, 82Q/127L/144Y/378S, 82Q/127L/235S, 82Q/127L/290Q, 82Q/144Y/266V/290Q/378S, 82Q/144Y/266V/378S, 82Q/235S/266V, 82Q/235S/266V/378S/383P, 82Q/235S/290V, 82Q/235S/378S, 82Q/266V, 82Q/266V/378S, 82Q/378S, 113A/270L/370S/384V, 115Q/256G/333L/375S, 115Q/256G/375S, 115Q/273V/375S, 115Q/375S, 127L/144Y/290V, 127L/149H/150Q, 127L/266V/290Q/378S, 127L/378S/383P, 144Y/235S/290Q, 144Y/266V/290Q, 233L/334L, 233L/376L, 235S, 235S/290Q, 256G/333L, 262L, 266V/378S, 270L/370S, 273V, 276E/352L, 290Q, 290V, 290V/378S, 333L, 358W, 370S, 375S, and 378S. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from A18D, A18D/S113A/K270L,
A18D/S113A/K270L/K329A/T370S, A18D/S259R/T370S/A384V, A18D/K270L, A18D/K270L/T370S, A18D/K270L/T370S/A384V, A18D/T370S, A18L, A18L/S113A/K270L, A18L/S113A/K270L/T370S/A384V, A18L/S113A/K329A/A384V, A18L/S113E/K270L/T370S/G412S, A18L/T370S, A18L/T370S/A384V, Q73H, Q73H/K115Q/C184A/R341 G, Q73H/C184A/R256G, Q73H/D273V/T333L/L375S, Q73H/T333L, Q73R, Q73R/K115Q, Q73R/K115Q/C184A/T333L, Q73R/K115Q/R256G, Q73R/C184A/D273V/T333L, Q73R/C184A/T333L, Q73R/R256G, Q73R/R256G/L375S, Q73R/D273V, Q73R/D273V/T333L/L375S, Q73R/D273V/L375S, Q73R/T333L, Q73R/T333L/L375S, Q73R/L375S, Q73R/G379V, E77V/A233L/I238V/R376L, E77V/A233L/G334L/R376L, E77V/V262L/R376L, E77V/Q276E, E77V/G334L/R376L, L82Q, L82Q/I127L, L82Q/I127L/W144Y/T235S/R290V, L82Q/I127L/W144Y/A266V, L82Q/I127L/W144Y/A266V/R290V, L82Q/I127L/W144Y/K378S, L82Q/I127L/T235S, L82Q/I127L/R290Q, L82Q/W144Y/A266V/R290Q/K378S, L82Q/W144Y/A266V/K378S, L82Q/T235S/A266V, L82Q/T235S/A266V/K378S/A383P, L82Q/T235S/R290V, L82Q/T235S/K378S, L82Q/A266V, L82Q/A266V/K378S, L82Q/K378S, S113A/K270L/T370S/A384V, K115Q/R256G/T333L/L375S,
K115Q/R256G/L375S, K115Q/D273V/L375S, K115Q/L375S, I127L/W144Y/R290V, I127L/D149H/H150Q, I127L/A266V/R290Q/K378S, 1127L/K378S/A383P, W144Y/T235S/R290Q, W144Y/A266V/R290Q, A233L/G334L, A233L/R376L, T235S, T235S/R290Q, R256G/T333L, V262L, A266V/K378S, K270L/T370S, D273V, Q276E/V352L, R290Q, R290V, R290V/K378S, T333L, I358W, T370S, L375S, and K378S.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from 18, 18/113, 18/113/252, 18/113/252/336/384, 18/113/252/370, 18/113/270, 18/113/270/370/384, 18/259/370/384, 18/270, 18/270/370, 18/270/370/384, 18/370, 18/370/384, 73, 73/115/184/341 , 73/115/341 , 73/131/256/273/333/341 /375, 73/184/256, 73/184/256/341 , 73/184/256/375, 73/184/273/333, 73/184/333, 73/184/341/375, 73/256, 73/256/273, 73/256/273/375, 73/256/333/375, 73/256/375, 73/273, 73/273/333/341 , 73/273/333/375, 73/273/375, 73/333, 73/333/341/375, 73/333/375, 73/341 , 73/375, 73/379, 77, 77/118, 77/118/143/262/276/334/376, 77/118/143/334/352/376, 77/118/143/376, 77/118/233, 77/118/233/352/376, 77/118/262/334/376, 77/118/334, 77/143/262/276, 77/143/262/334, 77/143/376, 77/233/238/376, 77/233/262, 77/233/276/376, 77/233/334/376, 77/262/376, 77/276, 77/334/376, 77/352, 82, 82/127, 82/127/144/235/290, 82/127/144/266, 82/127/144/266/290, 82/127/144/378, 82/127/235, 82/127/235/266/358, 82/127/290, 82/127/290/358, 82/127/290/378, 82/144/266/290/378, 82/144/266/378, 82/150/235/266, 82/235/266, 82/235/266/290, 82/235/266/378/383, 82/235/290, 82/235/378, 82/266, 82/266/378, 82/290, 82/378, 113, 113/252, 113/252/270/370, 113/270/370/384, 113/336, 118/233/276/334, 118/334, 127/235/266, 127/266/290/378, 127/378/383, 143, 143/276, 143/276/334, 144/235/290, 144/266/290, 144/290/358, 233/334, 233/376, 235, 235/266/290/358/383, 235/358, 252/329/336/370, 252/384, 256/333, 256/375, 262, 262/276, 262/334, 266/290, 266/378, 270/370, 273, 276/352, 290, 290/378, 333, 334, 334/376, 336/370, 341 , 358, 370, 375, and 378. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from 18D, 18D/113A/252A, 18D/113A/252A/336Q/384V, 18D/113A/270L, 18D/259R/370S/384V, 18D/270L, 18D/270L/370S, 18D/270L/370S/384V, 18D/370S, 18L, 18L/113A, 18L/113A/270L, 18L/113A/270L/370S/384V, 18L/113E, 18L/113E/252A/370S, 18L/370S, 18L/370S/384V, 73H, 73H/115Q/184A/341 G, 73H/115Q/341 G, 73H/184A/256G, 73H/256G/273V/375S, 73H/256G/333L/375S, 73H/273V/333L/341 G, 73H/273V/333L/375S, 73H/333L, 73R, 73R/115Q/341 G, 73R/131 D/256G/273V/333L/341 G/375S, 73R/184A/256G/341 G, 73R/184A/256G/375S, 73R/184A/273V/333L, 73R/184A/333L,
73R/184A/341 G/375S, 73R/256G, 73R/256G/273V, 73R/256G/273V/375S, 73R/256G/375S, 73R/273V, 73R/273V/333L/375S, 73R/273V/375S, 73R/333L, 73R/333L/341 G/375S, 73R/333L/375S, 73R/341 G, 73R/375S, 73R/379V, 77V, 77V/118S, 77V/118S/143H/262L/276E/334L/376L,
77V/118S/143H/334L/352L/376L, 77V/118S/143H/376L, 77V/118S/233L, 77V/118S/233L/352L/376L, 77V/118S/262L/334L/376L, 77V/118S/334L, 77V/143H/262L/276E, 77V/143H/262L/334L, 77V/143H/376L, 77V/233L/238V/376L, 77V/233L/262L, 77V/233L/276E/376L, 77V/233L/334L/376L, 77V/262L/376L, 77V/276E, 77V/334L/376L, 77V/352L, 82Q, 82Q/127L, 82Q/127L/144Y/235S/290V, 82Q/127L/144Y/266V, 82Q/127L/144 Y/266 V/290 V, 82Q/127L/144Y/378S, 82Q/127L/235S, 82Q/127L/235S/266V/358W, 82Q/127L/290Q, 82Q/127L/290Q/358W, 82Q/127L/290V/378S, 82Q/144Y/266V/290Q/378S, 82Q/144 Y/266 V/378S, 82Q/150Q/235S/266V, 82Q/235S/266V, 82Q/235S/266V/290Q, 82Q/235S/266V/378S/383P, 82Q/235S/290V, 82Q/235S/378S, 82Q/266V, 82Q/266V/378S, 82Q/290V, 82Q/378S, 113A/270L/370S/384V, 113E, 113E/252A,
113E/252A/270L/370S, 113E/336Q, 118S/233L/276E/334L, 118S/334L, 127L/235S/266V, 127L/266V/290Q/378S, 127L/378S/383P, 143H, 143H/276E, 143H/276E/334L, 144Y/235S/290Q, 144Y/266V/290Q, 144Y/290V/358W, 233L/334L, 233L/376L, 235S, 235S/266V/290Q/358W/383P, 235S/358W, 252A/329A/336Q/370S, 252A/384V, 256G/333L, 256G/375S, 262L, 262L/276E, 262L/334L, 266V/290Q, 266V/378S, 270L/370S, 273V, 276E/352L, 290Q, 290V/378S, 333L, 334L, 334L/376L, 336Q/370S, 341 G, 358W, 370S, 375S, and 378S. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2540 and one or more residue differences as compared to SEQ ID NO: 2540, selected from A18D, A18D/S113A/N252A, A18D/S113A/N252A/G336Q/A384V, A18D/S113A/K270L, A18D/S259R/T370S/A384V, A18D/K270L, A18D/K270L/T370S, A18D/K270L/T370S/A384V,
A18D/T370S, A18L, A18L/S113A, A18L/S113A/K270L, A18L/S113A/K270L/T370S/A384V, A18L/S113E, A18L/S113E/N252A/T370S, A18L/T370S, A18L/T370S/A384V, Q73H, Q73H/K115Q/C184A/R341 G, Q73H/K115Q/R341 G, Q73H/C184A/R256G, Q73H/R256G/D273V/L375S, Q73H/R256G/T333L/L375S, Q73H/D273V/T333L/R341 G, Q73H/D273V/T333L/L375S, Q73H/T333L, Q73R, Q73R/K115Q/R341 G, Q73R/G131 D/R256G/D273V/T333L/R341 G/L375S, Q73R/C184A/R256G/R341 G, Q73R/C184A/R256G/L375S, Q73R/C184A/D273V/T333L, Q73R/C184A/T333L,
Q73R/C184A/R341 G/L375S, Q73R/R256G, Q73R/R256G/D273V, Q73R/R256G/D273V/L375S, Q73R/R256G/L375S, Q73R/D273V, Q73R/D273V/T333L/L375S, Q73R/D273V/L375S, Q73R/T333L, Q73R/T333L/R341 G/L375S, Q73R/T333L/L375S, Q73R/R341 G, Q73R/L375S, Q73R/G379V, E77V, E77V/A118S, E77V/A118S/D143H/V262L/Q276E/G334L/R376L,
E77V/A118S/D143H/G334L/V352L/R376L, E77V/A118S/D143H/R376L, E77V/A118S/A233L, E77V/A118S/A233L/V352L/R376L, E77V/A118S/V262L/G334L/R376L, E77V/A118S/G334L, E77V/D143H/V262L/Q276E, E77V/D143H/V262L/G334L, E77V/D143H/R376L, E77V/A233L/I238V/R376L, E77V/A233L/V262L, E77V/A233L/Q276E/R376L, E77V/A233L/G334L/R376L, E77V/V262L/R376L, E77V/Q276E, E77V/G334L/R376L, E77V/V352L, L82Q, L82Q/I127L,
L82Q/I127L/W144Y/T235S/R290V, L82Q/I127L/W144Y/A266V, L82Q/I127L/W144Y/A266V/R290V, L82Q/I127L/W144Y/K378S, L82Q/I127L/T235S, L82Q/I127L/T235S/A266V/I358W, L82Q/I127L/R290Q, L82Q/I127L/R290Q/I358W, L82Q/I127L/R290V/K378S, L82Q/W144Y/A266V/R290Q/K378S, L82Q/W144Y/A266V/K378S, L82Q/H150Q/T235S/A266V, L82Q/T235S/A266V, L82Q/T235S/A266V/R290Q, L82Q/T235S/A266V/K378S/A383P, L82Q/T235S/R290V, L82Q/T235S/K378S, L82Q/A266V, L82Q/A266V/K378S, L82Q/R290V, L82Q/K378S,
S113A/K270L/T370S/A384V, S113E, S113E/N252A, S113E/N252A/K270L/T370S, S113E/G336Q, A118S/A233L/Q276E/G334L, A118S/G334L, 1127L/T235S/A266V, I127L/A266V/R290Q/K378S, I127L/K378S/A383P, D143H, D143H/Q276E, D143H/Q276E/G334L, W144Y/T235S/R290Q, W144Y/A266V/R290Q, W144Y/R290V/I358W, A233L/G334L, A233L/R376L, T235S, T235S/A266V/R290Q/I358W/A383P, T235S/I358W, N252A/K329A/G336Q/T370S, N252A/A384V, R256G/T333L, R256G/L375S, V262L, V262L/Q276E, V262L/G334L, A266V/R290Q, A266V/K378S, K270L/T370S, D273V, Q276E/V352L, R290Q, R290V/K378S, T333L, G334L, G334L/R376L, G336Q/T370S, R341 G, I358W, T370S, L375S, and K378S.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3402 and one or more residue differences as compared to SEQ ID NO: 3402, selected from 18, 18/77/118/169/233/238/256/266/270, 18/77/150/256/376, 18/118, 18/118/143/169/238/256/266/333, 18/118/150/169/184/238/256/270/378, 18/118/169, 18/118/233/238, 18/143, 18/143/256, 18/150/169, 18/150/266, 18/169/184/290/333/376, 18/169/233/333, 18/169/392, 18/184/270, 18/233/256/334, 18/256/266/290/378, 77/113/118/256/266/290, 77/118/290, 77/169/270/290, 77/233, 77/233/256, 77/238, 113/118/169/184/256/376, 113/169, 113/233, 118, 118/143/238, 118/233/238/256/378, 118/233/238/270, 143/169/233/270, 143/233/266/290/376, 143/238/270/290/378, 143/256/334, 143/290, 169, 169/256/266, 183/270/333, 233/238, 233/238/376, 233/392, 238/256, 256/290/303, 270, 290, 376, and 378. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3402 and one or more residue differences as compared to SEQ ID NO: 3402, selected from 18L, 18L/77V/118S/169T/233L/238V/256R/266V/270P, 18L/77V/150N/256R/376L, 18L/118S, 18L/118S/143H/169T/238V/256R/266V/333T, 18L/118S/150N/169T/184A/238V/256R/270P/378S, 18L/118S/169T, 18L/118S/233L/238V, 18L/143H, 18L/143H/256R, 18L/150N/169T, 18L/150N/266V, 18L/169T/184A/290K/333T/376L, 18L/169T/233L/333T, 18L/169T/392P, 18L/184A/270P, 18L/233L/256R/334L, 18L/256R/266V/290K/378S, 77V/113E/118S/256R/266V/290K, 77V/118S/290K, 77V/169T/270P/290K, 77V/233L, 77V/233L/256R, 77V/238V, 113E/118S/169T/184A/256R/376L, 113E/169T, 113E/233L, 118S, 118S/143H/238V, 118S/233L/238V/256R/378S, 118S/233L/238V/270P, 143H/169T/233L/270P, 143H/233L/266V/290K/376L, 143H/238V/270P/290K/378S, 143H/256R/334L, 143H/290K, 169T, 169T/256R/266V, 183L/270P/333T, 233U238V, 233L/238V/376L, 233L/392P, 238V/256R, 256R/290K/303G, 270P, 290K, 376L, and 378S. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3402 and one or more residue differences as compared to SEQ ID NO: 3402, selected from A18L, A18L/E77V/A118S/C169T/A233L/I238V/G256R/A266V/K270P, A18L/E77V/H150N/G256R/R376L, A18L/A118S,
A18L/A118S/D143H/C169T/I238V/G256R/A266V/L333T,
A18L/A118S/H150N/C169T/C184A/I238V/G256R/K270P/K378S, A18L/A118S/C169T, A18L/A118S/A233L/I238V, A18L/D143H, A18L/D143H/G256R, A18L/H150N/C169T, A18L/H150N/A266V, A18L/C169T/C184A/R290K/L333T/R376L, A18L/C169T/A233L/L333T, A18L/C169T/C392P, A18L/C184A/K270P, A18L/A233L/G256R/G334L,
A18L/G256R/A266V/R290K/K378S, E77V/S113E/A118S/G256R/A266V/R290K, E77V/A118S/R290K, E77V/C169T/K270P/R290K, E77V/A233L, E77V/A233L/G256R, E77V/I238V,
S113E/A118S/C169T/C184A/G256R/R376L, S113E/C169T, S113E/A233L, A118S, A118S/D143H/I238V, A118S/A233L/I238V/G256R/K378S, A118S/A233L/I238V/K270P, D143H/C169T/A233L/K270P, D143H/A233L/A266V/R290K/R376L, D143H/I238V/K270P/R290K/K378S, D143H/G256R/G334L, D143H/R290K, C169T, C169T/G256R/A266V, V183L/K270P/L333T, A233L/I238V, A233L/I238V/R376L, A233L/C392P, I238V/G256R, G256R/R290K/E303G, K270P, R290K, R376L, and K378S.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3402 and one or more residue differences as compared to SEQ ID NO: 3402, selected from 18, 18/77/118/169/266/334/376, 18/77/150/256/376, 18/77/256/290/334, 18/113/238, 18/118, 18/118/143/169/238/256/266/333, 18/118/143/169/376, 18/118/169, 18/118/233/238, 18/118/266, 18/143, 18/143/169/233/238/358, 18/143/233/238/290/376, 18/143/256, 18/143/290, 18/143/333/334/378, 18/150/169, 18/150/266, 18/169/184/290/333/376, 18/169/233/333, 18/169/238/270/334/376, 18/169/392, 18/233/256/334, 18/233/266/290/376, 18/256/266/290/378, 18/266/270/378, 18/378, 77/113/121/143/233/334, 77/118/169/233/266/270, 77/118/238/376, 77/118/290, 77/169/270/290, 77/233, 77/238, 113/238/376, 118, 118/143/238, 118/143/392, 118/169/184/233/266/270/333, 118/233/238/256/378, 118/256/290/333/334/376/378, 118/256/334/376, 118/266/290, 143/169/233/270, 143/233/266/290/376, 143/238/270/290/378, 143/256/334, 143/290, 143/364, 150/233/333, 169, 169/256/266, 183/270/333, 233/238, 233/238/376, 233/392, 238/256, 238/266/270/378, 256/266/334, 266/270/376, 266/333, and 378. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3402 and one or more residue differences as compared to SEQ ID NO: 3402, selected from 18L, 18L/77V/118S/169T/266V/334L/376L, 18L/77V/150N/256R/376L, 18L/77V/256R/290K/334L, 18L/113E/238V, 18L/118S, 18L/118S/143H/169T/238V/256R/266V/333T, 18L/118S/143H/169T/376L, 18L/118S/169T, 18L/118S/233L/238V, 18L/118S/266V, 18L/143H, 18L/143H/169T/233L/238V/358W, 18L/143H/233L/238V/290K/376L, 18L/143H/256R, 18L/143H/290K, 18L/143H/333T/334L/378S, 18L/150N/169T, 18L/150N/266V, 18L/169T/184A/290K/333T/376L, 18L/169T/233L/333T, 18L/169T/238V/270P/334L/376L, 18L/169T/392P, 18L/233L/256R/334L, 18L/233L/266V/290K/376L, 18L/256R/266V/290K/378S, 18L/266V/270P/378S, 18L/378S, 77V/113E/121 C/143H/233L/334L, 77V/118S/169T/233L/266 V/270 P, 77V/118S/238V/376L, 77V/118S/290K, 77V/169T/270P/290K, 77V/233L, 77V/238V, 113E/238V/376L, 118S, 118S/143H/238V, 118S/143H/392P, 118S/169T/184A/233L/266V/270P/333T, 118S/233L/238V/256R/378S,
118S/256R/290K/333T/334L/376L/378S, 118S/256R/334L/376L, 118S/266V/290K, 143H/169T/233L/270P, 143H/233L/266V/290K/376L, 143H/238V/270P/290K/378S, 143H/256R/334L, 143H/290K, 143H/364R, 150N/233L/333T, 169T, 169T/256R/266V, 183L/270P/333T, 233L/238V, 233L/238V/376L, 233L/392P, 238V/256R, 238V/266V/270P/378S, 256R/266V/334L, 266V/270P/376L, 266V/333T, and 378S. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3402 and one or more residue differences as compared to SEQ ID NO: 3402, selected from A18L, A18L/E77V/A118S/C169T/A266V/G334L/R376L, A18L/E77V/H150N/G256R/R376L, A18L/E77V/G256R/R290K/G334L, A18L/S113E/I238V, A18L/A118S,
A18L/A118S/D143H/C169T/I238V/G256R/A266V/L333T, A18UA118S/D143H/C169T/R376L,
A18L/A118S/C169T, A18L/A118S/A233L/I238V, A18L/A118S/A266V, A18L/D143H, A18L/D143H/C169T/A233L/I238V/I358W, A18L/D143H/A233L/I238V/R290K/R376L, A18L/D143H/G256R, A18L/D143H/R290K, A18L/D143H/L333T/G334L/K378S, A18L/H150N/C169T, A18L/H150N/A266V, A18L/C169T/C184A/R290K/L333T/R376L, A18L/C169T/A233L/L333T, A18L/C169T/I238V/K270P/G334L/R376L, A18L/C169T/C392P, A18L/A233L/G256R/G334L,
A18L/A233L/A266V/R290K/R376L, A18L/G256R/A266V/R290K/K378S, A18L/A266V/K270P/K378S, A18L/K378S, E77V/S113E/W121 C/D143H/A233L/G334L, E77V/A118S/C169T/A233L/A266V/K270P, E77V/A118S/I238V/R376L, E77V/A118S/R290K, E77V/C169T/K270P/R290K, E77V/A233L, E77V/I238V, S113E/I238V/R376L, A118S, A118S/D143H/I238V, A118S/D143H/C392P,
A118S/C169T/C184A/A233L/A266V/K270P/L333T, A118S/A233L/I238V/G256R/K378S,
A118S/G256R/R290K/L333T/G334L/R376L/K378S, A118S/G256R/G334L/R376L, A118S/A266V/R290K, D143H/C169T/A233L/K270P, D143H/A233L/A266V/R290K/R376L, D143H/I238V/K270P/R290K/K378S, D143H/G256R/G334L, D143H/R290K, D143H/Q364R, H150N/A233L/L333T, C169T, C169T/G256R/A266V, V183L/K270P/L333T, A233L/I238V, A233L/I238V/R376L, A233L/C392P, I238V/G256R, I238V/A266V/K270P/K378S, G256R/A266V/G334L, A266V/K270P/R376L, A266V/L333T, and K378S.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3662 and one or more residue differences as compared to SEQ ID NO: 3662, selected from 77/156, 77/156/270, 77/184/244, 77/184/333, 77/244/270, 77/270, 143, 143/169/238/257/344, 143/169/255, 143/169/255/257, 143/169/343, 143/255, 143/255/257, 143/255/344, 143/257, 156, 156/270, 169/238, 169/238/255/257/344, 169/257/344, 169/344, 184, 184/244/324, 238, 238/255/344, 244/270, 257/344, and 270. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3662 and one or more residue differences as compared to SEQ ID NO: 3662, selected from 77E/156L, 77E/156L/270P, 77E/184A/244K, 77E/184A/333T, 77E/244K/270P, 77E/270P, 77E/270R, 143E, 143E/169T/238V/257R/344K, 143E/169T/255R, 143E/169T/255R/257R, 143E/255R, 143E/255R/257R, 143E/255R/344K, 143E/257R, 143H/169T/343R, 156L, 156L/270R, 169T/238V, 169T/238V/255R/257R/344M, 169T/257R/344M, 169T/344K, 169T/344M, 184A, 184A/244K/324M, 238V, 238V/255R/344M, 244K/270P, 257R/344K, and 270P. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3662 and one or more residue differences as compared to SEQ ID NO: 3662, selected from V77E/E156L, V77E/E156L/K270P, V77E/C184A/V244K, V77E/C184A/L333T, V77E/V244K/K270P, V77E/K270P, V77E/K270R, D143E, D143E/C169T/I238V/K257R/E344K, D143E/C169T/A255R, D143E/C169T/A255R/K257R, D143E/A255R, D143E/A255R/K257R, D143E/A255R/E344K, D143E/K257R, D143H/C169T/E343R, E156L, E156L/K270R, C169T/I238V, C169T/I238V/A255R/K257R/E344M, C169T/K257R/E344M, C169T/E344K, C169T/E344M, C184A, C184A/V244K/V324M, I238V, I238V/A255R/E344M, V244K/K270P, K257R/E344K, and K270P.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3662 and one or more residue differences as compared to SEQ ID NO: 3662, selected from 77, 77/156/184/244/324, 77/184/270, 143, 143/238, 143/238/255, 143/238/255/343, 143/238/255/392, 143/238/344, 143/255, 143/257/392, 143/344, 169, 169/238/255/344, 169/238/344, 169/255, 169/343, 169/392, 238/344, 255, 255/257, 255/257/392, 257, and 344. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3662 and one or more residue differences as compared to SEQ ID NO: 3662, selected from 77E, 77E/156L/184A/244K/324M, 77E/184A/270P, 143E/238V, 143 E/238 V/255 R/343R, 143E/238V/255R/392P, 143E/238V/344M, 143E/257R/392P, 143E/344M, 143H, 143H/238V/255R, 143H/255R, 169T, 169T/238V/255R/344M, 169T/238V/344M, 169T/255R, 169T/343R, 169T/392P, 238V/344M, 255R, 255R/257R, 255R/257R/392P, 257R, and 344M. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3662 and one or more residue differences as compared to SEQ ID NO: 3662, selected from V77E, V77E/E156L/C184A/V244K/V324M, V77E/C184A/K270P, D143E/I238V, D143E/I238V/A255R/E343R, D143E/I238V/A255R/C392P, D143E/I238V/E344M, D143E/K257R/C392P, D143E/E344M, D143H, D143H/I238V/A255R, D143H/A255R, C169T, C169T/I238V/A255R/E344M, C169T/I238V/E344M, C169T/A255R, C169T/E343R, C169T/C392P, I238V/E344M, A255R, A255R/K257R, A255R/K257R/C392P, K257R, and E344M.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3788 and one or more residue differences as compared to SEQ ID NO: 3788, selected from 20, 22, 26, 42, 43, 44, 45, 50, 51 , 52, 53, 54, 55, 60, 62, 64, 65, 95, 97, 99, 103, 104, 111 , 112, 115, 115/143/169, 115/169/343, 115/343, 116, 116/343, 143, 143/169, 154/439, 169, 169/324, 169/324/343, 169/343, 186, 234, 236, 238, 238/246, 239, 239/244, 241 , 242, 243, 244, 250, 251 , 254, 255, 260, 283, 284, 295, 296, 298/326, 319, 322, 323, 324, 324/343, 326, 328, 329, 333, 334, 338, 340, 341 , 343, 344, 347, 366, 434, 439, 442, 443, 444, 445, 446, and 447. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3788 and one or more residue differences as compared to SEQ ID NO: 3788, selected from 20R, 22F, 22L, 22N, 22R, 22V, 22W, 26R, 42G, 42P, 43L, 43P, 43V, 44G, 44P, 44V, 45A, 45E, 45G, 45L, 45M, 45Q, 45R, 45W, 50R, 51 Q, 51 R, 52K, 52P, 52R, 52T, 53A, 53H, 53R, 53S, 54R, 54S, 54T, 54V, 55G, 55M, 55S, 55T, 55V, 60L, 62G, 64A, 64D, 64E, 64G, 64R, 64S, 64T, 64V, 65A, 65M, 95G, 95R, 95V, 97A, 97C, 99G, 99R, 103D, 103F, 103G, 103R, 103S, 103T, 103V, 104G, 104T, 111 V, 112R, 115E, 115E/169T/343R, 115G, 115L, 115P, 115Q, 115Q/143E/169T, 115Q/169T/343R, 115Q/343R, 115R, 115T, 115W, 116E/343R, 116S, 143E, 143E/169T, 154E/439S, 169T, 169T/324M, 169T/324M/343R, 169T/343R, 186A, 186P, 186Q, 186S, 234W, 236T, 236V, 236Y, 238L/246G, 238V, 239D, 239K/244F, 239L, 241 R, 242M, 243E, 243G, 243L, 243Q, 243W, 244C, 244G, 244T, 250A, 250S, 251 A, 2511, 251 L, 251 S, 254R, 254T, 255C, 255K, 255L, 255M, 255R, 260A, 260C, 260I, 260T, 283G, 283S, 283T, 284A, 284G, 284R, 295F, 295I, 295V, 296H, 298I/326A, 319E, 319K, 319R, 319W, 322G, 322S, 322T, 323H, 323T, 323V, 324M, 324M/343R, 326A, 326L, 326Q, 328Y, 329G, 329T, 329W, 333M, 333Q, 333R, 333W, 333Y, 334E, 334W, 338A, 340A, 341 K, 343G, 343M, 343R, 343V, 344G, 344H, 344K, 344M, 344Q, 344S, 344T, 344V, 347R, 366S, 434Y, 439A, 439G, 439S, 439Y, 442G, 442H, 442Q, 443C, 443I, 443P, 443S, 444Q, 445G, 445L, 445M, 445T, 445V, 446F, 446G, 446R, 447L, 447N, and 447T. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 3788 and one or more residue differences as compared to SEQ ID NO: 3788, selected from G20R, P22F, P22L, P22N, P22R, P22V, P22W, K26R, S42G, S42P, I43L, I43P, I43V, T44G, T44P, T44V, V45A, V45E, V45G, V45L, V45M, V45Q, V45R, V45W, S50R, K51 Q, K51 R, A52K, A52P, A52R, A52T, M53A, M53H, M53R, M53S, Q54R, Q54S, Q54T, Q54V, E55G, E55M, E55S, E55T, E55V, F60L, R62G, P64A, P64D, P64E, P64G, P64R, P64S, P64T, P64V, N65A, N65M, T95G, T95R, T95V, S97A, S97C, S99G, S99R, Q103D, Q103F, Q103G, Q103R, Q103S, Q103T, Q103V, V104G, V104T, L111 V, G112R, K115E, K115E/C169T/E343R, K115G, K115L, K115P, K115Q, K115Q/D143E/C169T, K115Q/C169T/E343R, K115Q/E343R, K115R, K115T, K115W, D116E/E343R, D116S, D143E, D143E/C169T, K154E/R439S, C169T, C169T/V324M, C169T/V324M/E343R, C169T/E343R, R186A, R186P, R186Q, R186S, Y234W, I236T, I236V, I236Y, I238L/S246G, I238V, P239D, P239K/V244F, P239L, K241 R, G242M, V243E, V243G, V243L, V243Q, V243W, V244C, V244G, V244T, R250A, R250S, K251 A, K251 I, K251 L, K251 S, P254R, P254T, A255C, A255K, A255L, A255M, A255R, L260A, L260C, L260I, L260T, A283G, A283S, A283T, E284A, E284G, E284R, L295F, L295I, L295V, K296H, V298I/E326A, T319E, T319K, T319R, T319W, W322G, W322S, W322T, R323H, R323T, R323V, V324M, V324M/E343R, E326A, E326L, E326Q, H328Y, K329G, K329T, K329W, L333M, L333Q, L333R, L333W, L333Y, G334E, G334W, E338A, L340A, R341 K, E343G, E343M, E343R, E343V, E344G, E344H, E344K, E344M, E344Q, E344S, E344T, E344V, N347R, R366S, F434Y, R439A, R439G, R439S, R439Y, I442G, I442H, I442Q, A443C, A443I, A443P, A443S, S444Q, W445G, W445L, W445M, W445T, W445V, E446F, E446G, E446R, A447L, A447N, and A447T.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4150 and one or more residue differences as compared to SEQ ID NO: 4150, selected from 26, 26/236, 26/236/260/333, 26/236/295, 26/260, 26/260/295, 26/260/295/333, 26/295/319, 26/333, 64, 64/244, 64/251/260/347, 64/260/295/333, 64/260/347, 103/115/255, 103/115/343, 103/115/343/344, 103/143/255/284/343/344, 103/169/343/344, 103/255/284, 103/343/344, 115, 115/143/255, 115/143/255/343, 115/143/284, 115/143/343, 115/143/344, 115/169, 115/169/343, 115/255, 115/255/344, 115/284, 115/284/343/344, 115/284/344, 115/343, 115/343/344, 115/344, 143, 143/169/255/284/343/344, 143/169/343/344, 143/255, 143/255/284/343/344, 143/255/343, 143/255/343/344, 143/255/344, 143/343/344, 169, 169/255, 169/255/284, 169/255/343, 169/284, 169/343, 169/344, 236, 236/244/333, 236/260, 236/347, 244, 244/251 , 244/251/319, 244/260, 244/295, 244/347, 251 , 251/260/295/347, 251/333, 255, 255/284, 255/343, 255/343/344, 255/344, 259, 260, 260/295, 260/333, 284, 284/343/344, 284/344, 295, 295/333, 333, 343, 343/344, 344, and 347. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4150 and one or more residue differences as compared to SEQ ID NO: 4150, selected from 26R, 26R/236T, 26R/236T/260I/333Y, 26R/236T/295I, 26R/260I, 26R/260I/295I, 26R/260I/295I/333Y, 26R/295I/319K, 26R/333Y, 64A, 64A/244T, 64A/251 A/260I/347R, 64A/260I/295I/333Y, 64A/260I/347R, 103G/115E/255K, 103G/115E/343M, 103G/115E/343M/344G, 103G/143E/255K/284G/343M/344G, 103G/169T/343M/344G, 103G/255K/284A, 103G/343M/344G, 115E, 115E/143E/255K, 115E/143E/344G, 115E/169T, 115E/255K, 115E/284A/344G, 115E/284G/343M/344G, 115E/343M, 115E/344G, 115T, 115T/143E/255K/343M, 115T/143E/284G, 115T/143E/343M, 115T/169T, 115T/169T/343M, 115T/255K, 115T/255K/344G, 115T/284G, 115T/343M/344G, 115T/344G, 143E, 143E/169T/255K/284G/343M/344G, 143E/169T/343M/344G, 143E/255K, 143E/255K/284A/343M/344G, 143E/255K/343M, 143E/255K/343M/344G, 143E/255K/344G, 143E/343M/344G, 169T, 169T/255K, 169T/255K/284G, 169T/255K/343M, 169T/284A, 169T/284G, 169T/343M, 169T/344G, 236T, 236T/244T/333Y, 236T/260I, 236T/347R, 244T, 244T/251A, 244T/251 A/319K, 244T/260I, 244T/295I, 244T/347R, 251 A, 251 A/260I/295I/347R, 251A/333Y, 255K, 255K/284G, 255K/343M, 255K/343M/344G, 255K/344G, 259I, 260I, 260I/295I, 260I/333Y, 284A, 284A/343M/344G, 284G, 284G/344G, 295I, 295I/333Y, 333Y, 343M, 343M/344G, 344G, and 347R. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4150 and one or more residue differences as compared to SEQ ID NO: 4150, selected from K26R, K26R/I236T, K26R/I236T/L260I/L333Y, K26R/I236T/L295I, K26R/L260I, K26R/L260I/L295I, K26R/L260I/L295I/L333Y, K26R/L295I/T319K, K26R/L333Y, P64A, P64A/V244T,
P64A/K251 A/L260I/N347R, P64A/L260I/L295I/L333Y, P64A/L260I/N347R, Q103G/K115E/A255K, Q103G/K115E/E343M, Q103G/K115E/E343M/E344G, Q103G/D143E/A255K/E284G/E343M/E344G, Q103G/C169T/E343M/E344G, Q103G/A255K/E284A, Q103G/E343M/E344G, K115E,
K115E/D143E/A255K, K115E/D143E/E344G, K115E/C169T, K115E/A255K, K115E/E284A/E344G, K115E/E284G/E343M/E344G, K115E/E343M, K115E/E344G, K115T, K115T/D143E/A255K/E343M, K115T/D143E/E284G, K115T/D143E/E343M, K115T/C169T, K115T/C169T/E343M, K115T/A255K, K115T/A255K/E344G, K115T/E284G, K115T/E343M/E344G, K115T/E344G, D143E,
D143E/C169T/A255K/E284G/E343M/E344G, D143E/C169T/E343M/E344G, D143E/A255K, D143E/A255K/E284A/E343M/E344G, D143E/A255K/E343M, D143E/A255K/E343M/E344G, D143E/A255K/E344G, D143E/E343M/E344G, C169T, C169T/A255K, C169T/A255K/E284G, C169T/A255K/E343M, C169T/E284A, C169T/E284G, C169T/E343M, C169T/E344G, I236T, I236T/V244T/L333Y, I236T/L260I, I236T/N347R, V244T, V244T/K251A, V244T/K251 A/T319K, V244T/L260I, V244T/L295I, V244T/N347R, K251 A, K251 A/L260I/L295I/N347R, K251A/L333Y, A255K, A255K/E284G, A255K/E343M, A255K/E343M/E344G, A255K/E344G, S259I, L260I, L260I/L295I, L260I/L333Y, E284A, E284A/E343M/E344G, E284G, E284G/E344G, L295I, L295I/L333Y, L333Y, E343M, E343M/E344G, E344G, and N347R.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4278 and one or more residue differences as compared to SEQ ID NO: 4278, selected from 115, 115/117, 115/117/118, 115/117/118/152, 115/117/118/169, 115/117/118/246, 115/117/152, 115/117/152/169, 115/117/152/169/246, 115/117/152/246, 115/117/169, 115/117/246, 115/118, 115/118/152, 115/118/152/169, 115/118/152/169/246, 115/118/169, 115/118/246, 115/152, 115/152/169, 115/152/169/246, 115/152/246, 115/152/246/374, 115/169, 115/169/246, 115/246, 117, 117/118, 117/118/152, 117/118/152/169, 117/118/152/246, 117/118/169, 117/118/246, 117/152, 117/152/169, 117/152/246/288, 117/169, 117/246, 118/246, 152, 152/169, 152/169/246, 152/246, 169, 169/246, and 246. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4278 and one or more residue differences as compared to SEQ ID NO: 4278, selected from 115E, 115E/117A, 115E/117A/118T, 115E/117A/118T/152R, 115E/117A/118T/169T, 115E/117A/118T/246D, 115E/117A/152R, 115E/117A/152R/169T, 115E/117A/152R/169T/246D, 115E/117A/152R/246D, 115E/117A/169T, 115E/117A/246D, 115E/118T, 115E/118T/152R, 115E/118T/152R/169T, 115E/118T/152R/169T/246D, 115E/118T/169T, 115E/118T/246D, 115E/152R, 115E/152R/169T, 115E/152R/169T/246D, 115E/152R/246D, 115E/152R/246D/374C, 115E/169T, 115E/169T/246D, 115E/246D, 117A, 117A/118T, 117A/118T/152R, 117A/118T/152R/169T, 117A/118T/152R/246D, 117A/118T/169T, 117A/118T/246D, 117A/152R, 117A/152R/169T, 117A/152R/246D/288G, 117A/169T, 117A/246D, 118T/246D, 152R, 152R/169T, 152R/169T/246D, 152R/246D, 169T, 169T/246D, and 246D. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4278 and one or more residue differences as compared to SEQ ID NO: 4278, selected from K115E, K115E/K117A, K115E/K117A/S118T, K115E/K117A/S118T/T152R, K115E/K117A/S118T/C169T, K115E/K117A/S118T/S246D, K115E/K117A/T152R, K115E/K117A/T152R/C169T,
K115E/K117A/T152R/C169T/S246D, K115E/K117A/T152R/S246D, K115E/K117A/C169T, K115E/K117A/S246D, K115E/S118T, K115E/S118T/T152R, K115E/S118T/T152R/C169T, K115E/S118T/T152R/C169T/S246D, K115E/S118T/C169T, K115E/S118T/S246D, K115E/T152R, K115E/T152R/C169T, K115E/T152R/C169T/S246D, K115E/T152R/S246D,
K115E/T152R/S246D/Y374C, K115E/C169T, K115E/C169T/S246D, K115E/S246D, K117A,
K117A/S118T, K117A/S118T/T152R, K117A/S118T/T152R/C169T, K117A/S118T/T152R/S246D, K117A/S118T/C169T, K117A/S118T/S246D, K117A/T152R, K117A/T152R/C169T,
K117A/T152R/S246D/S288G, K117A/C169T, K117A/S246D, S118T/S246D, T152R, T152R/C169T, T152R/C169T/S246D, T152R/S246D, C169T, C169T/S246D, and S246D.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4278 and one or more residue differences as compared to SEQ ID NO: 4278, selected from 73, 101 , 114, 115, 118, 143, 145, 150, 151 , 152, 153, 154, 156, 157, 160, 161 , 163, 166, 167, 168, 169, 171 , 172, 190, 191 , 192, 195, 213, 214, 218, 219, 223, 225, 233, 233/343, 234, 235, 236, 237, 238, 239, 239/250, 243, 244, 249, 251 , 254, 255, 257, 258, 259, 261 , 262, 262/283, 277/292, 283, 284, 286, 289, 292, 295, and 296. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4278 and one or more residue differences as compared to SEQ ID NO: 4278, selected from 73L, 73R, 101 S, 114S, 115A, 115Q, 115R, 115S, 118R, 143E, 143L, 143Q, 143V, 145Y, 1501, 150L, 150M, 150T, 151 A, 151 D, 151 F, 151 H, 151 R, 152L, 153G, 154P, 154S, 154Y, 156D, 156L, 156Q, 156W, 157L,
157P, 157R, 160R, 160T, 160V, 161 R, 163K, 163L, 163Q, 163R, 166T, 167Q, 167R, 167T, 168A, 168N,
168V, 169L, 169V, 1711, 171 L, 171 M, 172L, 172R, 172V, 190F, 191 H, 191 L, 191 V, 192E, 192G, 192R, 192S, 195L, 195M, 195T, 195V, 213R, 214L, 214V, 218P, 219R, 223A, 223D, 223Q, 225V, 233F, 233G,
233L, 233Q, 233R, 233T/343G, 233W, 234L, 234V, 235C, 235D, 235E, 235S, 236M, 236T, 237Q, 238L,
238V, 239A, 239E, 239N/250H, 239T, 243I, 243Q, 243V, 244A, 244F, 244L, 244S, 244T, 244W, 249G, 249I, 249L, 249R, 249S, 249T, 251 H, 251 Q, 254A, 254E, 255G, 255S, 255W, 257L, 257T, 258V, 259L, 259N, 261 A, 261 K, 261 R, 261 T, 261 V, 262I/283V, 262L, 277S/292V, 283G, 283T, 284I, 284L, 284R, 284S, 286E, 286G, 286I, 286L, 286V, 289L, 292E, 292G, 295V, 296C, and 296R. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4278 and one or more residue differences as compared to SEQ ID NO: 4278, selected from Q73L, Q73R, K101 S, V1 14S, K1 15A, K1 15Q, K1 15R, K1 15S, S1 18R, D143E, D143L, D143Q, D143V, W145Y, H150I, H150L, H150M, H150T, P151 A, P151 D, P151 F, P151 H, P151 R, T152L, D153G, K154P, K154S, K154Y, E156D, E156L, E156Q, E156W, K157L, K157P, K157R, A160R, A160T, A160V, K161 R, A163K, A163L, A163Q, A163R, S166T, H167Q, H167R, H167T, K168A, K168N, K168V, C169L, C169V, Q171 1, Q171 L, Q171 M, 1172L, 1172R, 1172V, 1190F, Q191 H, Q191 L, Q191 V, A192E, A192G, A192R, A192S, Q195L, Q195M, Q195T, Q195V, K213R, I214L, 1214V, E218P, L219R, E223A, E223D, E223Q, L225V, A233F, A233G, A233L, A233Q, A233R, A233T/E343G, A233W, Y234L, Y234V, T235C, T235D, T235E, T235S, I236M, I236T, D237Q, I238L, I238V, P239A, P239E, P239N/R250H, P239T, G243I, G243Q, G243V, V244A, V244F, V244L, V244S, V244T, V244W, K249G, K249I, K249L, K249R, K249S, K249T, K251 H, K251 Q, P254A, P254E, A255G, A255S, A255W, K257L, K257T, A258V, S259L, S259N, S261 A, S261 K, S261 R, S261 T, S261 V, V262I/A283V, V262L, P277S/M292V, A283G, A283T, E284I, E284L, E284R, E284S, T286E, T286G, T286I, T286L, T286V, P289L, M292E, M292G, I295V, K296C, and K296R.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4642 and one or more residue differences as compared to SEQ ID NO: 4642, selected from 93, 1 13, 1 13/1 14, 1 13/1 15, 1 13/143, 1 15/1 18, 1 15/177, 1 18/143/163/168/195/255/315, 1 18/143/255, 1 18/163/168/214/255/315, 1 18/168/214/315, 1 18/255, 124, 126, 129, 134, 143/154/255, 143/156/168/216/255, 143/163, 143/163/255, 143/168, 143/168/169/255/315, 143/255, 148, 156/214/315, 159, 163, 163/168, 163/168/169, 163/168/169/255/315, 163/168/169/315, 163/168/195/214/255/315, 163/214, 163/255, 163/255/315, 168/315, 186, 194, 197, 206, 207, 208, 210, 213, 255/315, 277, 293, 294, 315, 317, 320, 322, 328, 329, 330, 333, 352, 366, 392, and 445. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4642 and one or more residue differences as compared to SEQ ID NO: 4642, selected from 93A, 93F, 93G, 93H, 93M, 93S, 93T, 1 13C/1 15K, 1 13G/1 15K, 1 13S, 1 13S/1 14G, 1 13S/1 15E, 1 13S/143K, 113S/143R, 1 15K/1 18C, 1 15K/1 18G, 1 15R/177V, 1 18T/143Q/163V/168N/195E/255K/315L, 1 18T/143Q/255K, 1 18T/163V/168N/214L/255K/315L, 1 18T/168N/214L/315L, 1 18T/255K, 124T, 126V, 129M, 134R, 134V, 143Q/154L/255K, 143Q/156D/168N/216G/255K, 143Q/163V, 143Q/163V/255K, 143Q/168N, 143Q/168N/169L/255K/315L, 143Q/255K, 148L, 148Q, 148R, 156D/214L/315L, 159T, 163V, 163V/168M, 163V/168M/169L, 163V/168M/169T, 163V/168M/169V, 163V/168M/195E/214L/255K/315L, 163V/168N/169T/255K/315L, 163V/168N/169V/315L, 163V/214L, 163V/255K, 163V/255K/315L, 168N/315L, 186K, 194M, 197L, 206L, 206M, 207M, 208G, 21 OR, 213R, 255K/315L, 277D, 277G, 277S, 277W, 293G, 293P, 293R, 293S, 293T, 294G, 294R, 294V, 315L, 317A, 317C, 317G, 317L, 317Q, 317S, 320C, 320I, 320V, 320Y, 322F, 328F, 329L, 329S, 330S, 330T, 333G, 333Q, 333R, 352G, 352I, 366A, 366K, 392A, 445R, and 445Y. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 2 and one or more residue differences as compared to SEQ ID NO: 4642, selected from D93A, D93F, D93G, D93H, D93M, D93S, D93T, E1 13C/Q1 15K, E1 13G/Q1 15K, E1 13S, E1 13S/V1 14G, E1 13S/Q1 15E, E1 13S/D143K, E1 13S/D143R, Q115K/S1 18C, Q1 15K/S1 18G, Q1 15R/M177V, S1 18T/D143Q/A163V/K168N/Q195E/A255K/I315L, S1 18T/D143Q/A255K, S1 18T/A163V/K168N/I214L/A255K/I315L, S1 18T/K168N/I214L/I315L, S1 18T/A255K, H124T, T126V, L129M, T134R, T134V, D143Q/K154L/A255K, D143Q/E156D/K168N/D216G/A255K, D143Q/A163V, D143Q/A163V/A255K, D143Q/K168N, D143Q/K168N/C169L/A255K/I315L, D143Q/A255K, P148L, P148Q, P148R, E156D/I214L/I315L, S159T, A163V, A163V/K168M, A163V/K168M/C169L, A163V/K168M/C169T, A163V/K168M/C169V, A163V/K168M/Q195E/I214L/A255K/I315L, A163V/K168N/C169T/A255K/I315L, A163V/K168N/C169V/I315L, A163V/I214L, A163V/A255K, A163V/A255K/I315L, K168N/I315L, R186K, L194M, K197L, V206L, V206M, R207M, A208G, H210R, K213R, A255K/I315L, P277D, P277G, P277S, P277W, E293G, E293P, E293R, E293S, E293T, P294G, P294R, P294V, 1315L, M317A, M317C, M317G, M317L, M317Q, M317S, A320C, A320I, A320V, A320Y, W322F, H328F, K329L, K329S, A330S, A330T, L333G, L333Q, L333R, V352G, V352I, R366A, R366K, C392A, W445R, and W445Y.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4642 and one or more residue differences as compared to SEQ ID NO: 4642, selected from 127, 128, 129, 140, 180, 182, 194, 277, 293, 294, 299, 300, 317, 319, 320, 324, 333, 356, 366, and 392. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4642 and one or more residue differences as compared to SEQ ID NO: 4642, selected from 127L, 128V, 129M, 140V, 180L, 182Q, 182T, 194M, 277A, 277G, 277H, 277S, 293G, 293K, 293P, 293S, 294G, 294L, 294Q, 299T, 300T, 317A, 317F, 317I, 317S, 319A, 320I, 320Y, 324I, 333D, 333T, 356G, 366K, 392H, and 392M. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4642 and one or more residue differences as compared to SEQ ID NO: 4642, selected from I127L, L128V, L129M, 1140V, V180L, S182Q, S182T, L194M, P277A, P277G, P277H, P277S, E293G, E293K, E293P, E293S, P294G, P294L, P294Q, L299T, L300T, M317A, M317F, M317I, M317S, T319A, A320I, A320Y, V324I, L333D, L333T, A356G, R366K, C392H, and C392M.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4984 and one or more residue differences as compared to SEQ ID NO: 4984, selected from 1 13, 1 17, 1 18, 151 , 191 , 196, and 205. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4984 and one or more residue differences as compared to SEQ ID NO: 4984, selected from 113A, 113C, 113K, 113R, 113S, 113T, 117R, 118A, 118K, 118R, 151 A, 151C, 151 E, 151 F, 151G, 151 H, 1511, 151 K, 151 L, 151 M, 151 N, 1510, 151 R, 151 S, 151T, 151V, 151W, 151 Y, 191G, 191 K, 191 L, 191 M, 191 R, 191S, 191V, 196A, 196F, 196G, 196H, 1961, 196K, 196L, 196M, 196R, 196S, 196V, 196W, 196Y, and 205T. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4984 and one or more residue differences as compared to SEQ ID NO: 4984, selected from E113A, E113C, E113K, E113R, E113S, E113T, K117R, S118A, S118K, S118R, P151A, P151C, P151E, P151F, P151G, P151H, P151I, P151K, P151L, P151M, P151N, P151Q, P151R, P151S, P151T, P151V, P151W, P151Y, Q191G, Q191K, Q191 L, Q191M, Q191R, Q191S, Q191V, D196A, D196F, D196G, D196H, D196I, D196K, D196L, D196M, D196R, D196S, D196V, D196W, D196Y, and V205T.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4984 and one or more residue differences as compared to SEQ ID NO: 4984, selected from 114/115/143, 114/115/143/233, 114/115/233, 114/115/343, 114/143, 114/143/151/233/238/261, 114/143/213/261/343, 114/143/233, 114/143/233/238, 114/151/261/343, 114/213/238, 114/343, 115, 115/143, 115/143/151/154/213/261, 115/143/151/154/233/343, 115/143/151/233/261 , 115/143/151/233/343, 115/143/154/213/233/238/261 , 115/143/213, 115/143/233, 115/143/343, 115/151/154/233, 115/151/261, 115/213/233, 115/213/233/238, 115/213/261, 115/233/238, 115/238, 115/343, 115/348, 143, 143/151/154, 143/151/154/213, 143/154, 143/213/233, 143/213/233/238/261/343, 143/213/261/343, 143/233/261, 143/261, 151/154, 151/154/213/343, 151/154/343, 151/213, 151/213/233, 213/233/343, and 233/238/343. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4984 and one or more residue differences as compared to SEQ ID NO: 4984, selected from 114G/115E/143E/233T, 114G/115E/143V, 114G/115E/233T, 114G/115E/343G, 114G/143E/233Q, 114G/143L/233F/238V, 114G/143V,
114G/143V/151 R/233T/238 V/261 R, 114G/143V/213R/261 R/343G, 114G/151 R/261 R/343G, 114G/213R/238V, 114G/343G, 115E, 115E/143E, 115E/143E/213R, 115E/143L/151 R/154S/213R/261 R, 115E/143L/151 R/154S/233Q/343G, 115E/143L/151 R/233F/343G,
115E/143L/154S/213R/233Q/238V/261 R, 115E/143L/233Q, 115E/143L/233R, 115E/143L/343G, 115E/143V/151 R/233F/261 R, 115E/151 R/154S/233F, 115E/151 R/261 R, 115E/213R/233F/238V, 115E/213R/233R, 115E/213R/261 R, 115E/233F/238V, 115E/238V, 115E/343G, 115E/348F, 143E, 143E/151 R/154S, 143E/151 R/154S/213R, 143E/213R/233F/238V/261 R/343G, 143E/233F/261 R, 143L/213R/233R, 143L/261R, 143V/154S, 143V/213R/233R, 143V/213R/261 R/343G, 151 R/154S, 151 R/154S/213R/343G, 151 R/154S/343G, 151 R/213R, 151 R/213R/233F, 213R/233R/343G, and 233T/238V/343G. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 4984 and one or more residue differences as compared to SEQ ID NO: 4984, selected from
V114G/Q115E/D143E/A233T, V114G/Q115E/D143V, V114G/Q115E/A233T, V114G/Q115E/E343G, V114G/D143E/A233Q, V114G/D143L/A233F/I238V, V114G/D143V,
V114G/D143V/P151 R/A233T/I238V/S261 R, V114G/D143V/K213R/S261 R/E343G,
V114G/P151 R/S261 R/E343G, V114G/K213R/I238V, V114G/E343G, Q115E, Q115E/D143E, Q115E/D143E/K213R, Q115E/D143L/P151 R/K154S/K213R/S261 R,
Q115E/D143L/P151 R/K154S/A233Q/E343G, Q115E/D143L/P151 R/A233F/E343G,
Q115E/D143L/K154S/K213R/A233Q/I238V/S261 R, Q115E/D143L/A233Q, Q115E/D143L/A233R, Q115E/D143L/E343G, Q115E/D143V/P151 R/A233F/S261 R, Q115E/P151 R/K154S/A233F,
Q115E/P151 R/S261 R, Q115E/K213R/A233F/I238V, Q115E/K213R/A233R, Q115E/K213R/S261 R, Q115E/A233F/I238V, Q115E/I238V, Q115E/E343G, Q115E/L348F, D143E, D143E/P151 R/K154S, D143E/P151 R/K154S/K213R, D143E/K213R/A233F/I238V/S261 R/E343G, D143E/A233F/S261 R, D143L/K213R/A233R, D143L/S261 R, D143V/K154S, D143V/K213R/A233R,
D143V/K213R/S261 R/E343G, P151 R/K154S, P151 R/K154S/K213R/E343G, P151 R/K154S/E343G, P151 R/K213R, P151 R/K213R/A233F, K213R/A233R/E343G, and A233T/I238V/E343G.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5196 and one or more residue differences as compared to SEQ ID NO: 5196, selected from 117, 117/118/143/168/169/392, 117/118/143/392, 117/118/168/169, 117/118/168/169/392, 117/143/168/169/170/392, 117/143/168/169/392, 117/168/169, 117/168/169/392, 117/168/392, 117/169, 117/392, 118/143, 118/143/168/169, 118/143/169, 118/143/392, 118/168, 118/168/392, 118/169/392, 118/392, 143/168/169/392, 143/169, 143/392, 168/169, 168/169/392, 168/392, 169/392, and 392. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5196 and one or more residue differences as compared to SEQ ID NO: 5196, selected from 117R, 117R/118R/143Q/168K/169T/392A, 117R/118R/143Q/392A, 117R/118R/168K/169M, 117R/118R/168K/169T/392A, 117R/118R/168K/169T/392P, 117R/143Q/168K/169T/170H/392A, 117R/143Q/168K/169T/392A, 117R/168K/169T, 117R/168K/169T/392P, 117R/168K/392A, 117R/169T, 117R/392P, 118R/143Q, 118R/143Q/168K/169T, 118R/143Q/169T, 118R/143Q/392A, 118R/143Q/392P, 118R/168K, 118R/168K/392P, 118R/169T/392A, 118R/392A, 118R/392P, 143Q/168K/169T/392P, 143Q/169T, 143Q/392P, 168K/169T, 168K/169T/392A, 168K/169T/392P, 168K/392A, 168K/392P, 169T/392S, 392A, and 392P. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5196 and one or more residue differences as compared to SEQ ID NO: 5196, selected from K117R, K117R/S118R/D143Q/M168K/V169T/C392A, K117R/S118R/D143Q/C392A, K117R/S118R/M168K/V169M, K117R/S118R/M168K/V169T/C392A,
K117R/S118R/M168K/V169T/C392P, K117R/D143Q/M168K/V169T/R170H/C392A, K1 17R/D143Q/M168K/V169T/C392A, K1 17R/M168K/V169T, K117R/M168K/V169T/C392P,
K1 17R/M168K/C392A, K1 17R/V169T, K1 17R/C392P, S1 18R/D143Q, S1 18R/D143Q/M168K/V169T, S1 18R/D143Q/V169T, S1 18R/D143Q/C392A, S1 18R/D143Q/C392P, S1 18R/M168K, S1 18R/M168K/C392P, S1 18R/V169T/C392A, S1 18R/C392A, S1 18R/C392P, D143Q/M168K/V169T/C392P, D143Q/V169T, D143Q/C392P, M168K/V169T, M168K/V169T/C392A, M168K/V169T/C392P, M168K/C392A, M168K/C392P, V169T/C392S, C392A, and C392P.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5286 and one or more residue differences as compared to SEQ ID NO: 5286, selected from 134, 134/182/194/294, 134/182/277/293, 134/182/293/320, 134/182/294, 134/194/277, 134/194/277/317, 134/194/293, 134/194/293/294, 134/194/293/317, 134/194/294, 134/194/317, 134/194/317/320, 134/317, 182, 182/194, 182/194/293/320, 182/293/294, 194, 194/277/293/317, 194/277/317/320, 194/277/320, 194/293/294/317, 194/293/320, 277, 293, 317, 317/320, and 320. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5286 and one or more residue differences as compared to SEQ ID NO: 5286, selected from 134P, 134R/182T/277W/293G, 134R/182T/294Q, 134R/194M/277W, 134R/194M/277W/317S, 134R/194M/293P, 134R/194M/293P/294Q, 134R/194M/293P/317S,
134R/194M/294Q, 134R/194M/317S, 134R/194M/317S/320Y, 134R/317S, 134V, 134V/182T/194M/294Q, 134V/182T/293P/320Y, 134V/194M/277W, 134V/194M/293P, 182T, 182T/194M, 182T/194M/293G/320Y, 182T/293G/294Q, 194M, 194M/277W/293G/317S, 194M/277W/317S/320Y, 194M/277W/320Y, 194M/293P/294Q/317S, 194M/293P/320Y, 277W, 293P, 317S, 317S/320Y, and 320Y. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5286 and one or more residue differences as compared to SEQ ID NO: 5286, selected from T134P, T134R/S182T/P277W/E293G, T134R/S182T/P294Q, T134R/L194M/P277W, T134R/L194M/P277W/M317S, T134R/L194M/E293P, T134R/L194M/E293P/P294Q, T134R/L194M/E293P/M317S, T134R/L194M/P294Q, T134R/L194M/M317S, T134R/L194M/M317S/A320Y, T134R/M317S, T134V,
T134V/S182T/L194M/P294Q, T134V/S182T/E293P/A320Y, T134V/L194M/P277W, T134V/L194M/E293P, S182T, S182T/L194M, S182T/L194M/E293G/A320Y, S182T/E293G/P294Q, L194M, L194M/P277W/E293G/M317S, L194M/P277W/M317S/A320Y, L194M/P277W/A320Y, L194M/E293P/P294Q/M317S, L194M/E293P/A320Y, P277W, E293P, M317S, M317S/A320Y, and A320Y.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5412 and one or more residue differences as compared to SEQ ID NO: 5412, selected from 12/134, 13/134, 13/134/210, 22/134/152, 22/134/235/344, 64/93/134/235, 64/134/344/347, 77/134, 79/134/210, 84/134, 86/134, 87/134, 89/134, 93/134/152/235/344, 93/134/235/344, 93/134/244/344, 93/134/344, 131 , 134/152/344, 134/175, 134/210/382, 134/210/402, 134/210/456, 134/210/478, 134/344, 134/377, 134/378, 134/380, 134/381 , 134/382, 134/384, 134/386, 134/387, 134/388, 134/390, 134/391 , 134/393, 134/395, 134/398, 134/401 , 134/402, 134/406, 134/407, 134/421 , 134/456, 134/457, 134/459, 134/460, 134/464, 134/467, 134/468, 134/470, 134/471 , 134/473, 134/473/474, 134/474, and 134/475. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5412 and one or more residue differences as compared to SEQ ID NO: 5412, selected from 12N/134R, 13C/134T/210R, 13L/134R, 13L/134T, 13R/134R, 13S/134R, 13T/134R, 13V/134R, 13V/134T/210R, 22R/134R/152P, 22R/134R/235S/344T, 64R/93T/134R/235S, 64R/134R/344Q/347R, 77Y/134R, 79V/134T/21 OR, 84V/134R, 86S/134R, 87G/134R, 87M/134R, 89C/134R, 89L/134R, 93T/134R/152P/235S/344T, 93T/134R/235S/344T, 93T/134R/244L/344T, 93T/134R/344T, 1311, 134R/152P/344Q, 134R/175C, 134R/210R/402I, 134R/344T, 134R/377I, 134R/378R, 134R/378V, 134R/380V, 134R/381 D, 134R/382G, 134R/382K, 134R/382W, 134R/384F, 134R/386W, 134R/387G, 134R/387I, 134R/388F, 134R/390V, 134R/391 H, 134R/391 R, 134R/391T, 134R/393T, 134R/395I, 134R/395M, 134R/398S, 134R/401 V, 134R/402L, 134R/402V, 134R/406C, 134R/407V, 134R/421 V, 134R/456D, 134R/456T, 134R/457M, 134R/459L, 134R/460E, 134R/464A, 134R/467L, 134R/467R, 134R/467T, 134R/468S, 134R/470L, 134R/470P, 134R/471 M, 134R/471 S, 134R/473D/474I, 134R/473G, 134R/474T, 134R/475G, 134T/210R/382R, 134T/210R/456C, 134T/210R/478D, and 134T/380V. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5412 and one or more residue differences as compared to SEQ ID NO: 5412, selected from E12N/P134R, H13C/P134T/H210R, H13L/P134R, H13L/P134T, H13R/P134R, H13S/P134R, H13T/P134R, H13V/P134R, H13V/P134T/H210R, P22R/P134R/T152P, P22R/P134R/T235S/E344T, P64R/D93T/P134R/T235S, P64R/P134R/E344Q/N347R, E77Y/P134R, T79V/P134T/H210R, Q84V/P134R, Y86S/P134R, P87G/P134R, P87M/P134R, I89C/P134R, I89L/P134R, D93T/P134R/T152P/T235S/E344T, D93T/P134R/T235S/E344T, D93T/P134R/V244L/E344T, D93T/P134R/E344T, G131 I, P134R/T152P/E344Q, P134R/D175C, P134R/H210R/K402I, P134R/E344T, P134R/K377I, P134R/K378R, P134R/K378V, P134R/L380V, P134R/V381 D, P134R/D382G, P134R/D382K, P134R/D382W, P134R/A384F, P134R/E386W, P134R/L387G, P134R/L387I, P134R/E388F, P134R/Q390V, P134R/S391 H, P134R/S391 R, P134R/S391T, P134R/A393T, P134R/V395I, P134R/V395M, P134R/E398S, P134R/W401 V, P134R/K402L, P134R/K402V, P134R/K406C, P134R/M407V, P134R/L421 V, P134R/Q456D, P134R/Q456T, P134R/S457M, P134R/V459L, P134R/V460E, P134R/V464A, P134R/E467L, P134R/E467R, P134R/E467T, P134R/M468S, P134R/A470L, P134R/A470P, P134R/D471 M, P134R/D471 S, P134R/E473D/L474I, P134R/E473G, P134R/L474T, P134R/L475G, P134T/H210R/D382R, P134T/H210R/Q456C, P134T/H210R/S478D, and P134T/L380V. In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5412 and one or more residue differences as compared to SEQ ID NO: 5412, selected from 73/127/134/160/172/198/290, 73/127/134/160/198/333, 73/127/134/160/210/266, 73/127/134/172/192/266/333, 73/127/134/172/198, 73/127/134/210/290, 73/127/134/210/333, 73/127/134/290/333, 73/127/134/333, 73/134, 73/134/160/172/192/290, 73/134/160/172/198/333, 73/134/160/172/290, 73/134/160/266, 73/134/160/333, 73/134/172, 73/134/172/210/333, 73/134/172/266/333, 73/134/198/210, 73/134/198/210/266, 73/134/210/333, 73/134/333, 73/134/366, 127/134, 127/134/160/172/192/333, 127/134/160/172/210/266/290, 127/134/172/198/266/333, 127/134/192/210, 127/134/198/210/290, 127/134/210/266/290, 127/134/210/333, 134, 134/160/172/333, 134/160/198/266/290, 134/172/192/290/333, 134/192/198, 134/198, 134/198/266/333, 134/210, 134/210/290, and 134/333. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5412 and one or more residue differences as compared to SEQ ID NO: 5412, selected from 73K/127L/134R/160G/172R/198Q/290R, 73K/127L/134R/160G/198Q/333T, 73K/127L/134R/172R/192S/266A/333T, 73K/127L/134R/172R/198Q, 73K/127L/134R/333T, 73K/127L/134T/160G/210R/266A, 73K/127L/134T/210R/290R, 73K/127L/134T/210R/333T, 73K/127L/134T/290R/333T, 73K/134R, 73K/134R/160G/172R/290R, 73K/134R/160G/333T, 73K/134R/172R, 73K/134R/172R/210R/333T, 73K/134R/172R/266A/333T, 73K/134R/333T, 73K/134R/366H, 73K/134T/160G/172R/192S/290R, 73K/134T/160G/172R/198Q/333T, 73K/134T/160G/266A, 73K/134T/198Q/210R, 73K/134T/198Q/210R/266A, 73K/134T/210R/333T, 127L/134R, 127L/134R/160G/172R/192S/333T, 127L/134R/172R/198Q/266A/333T, 127L/134R/210R/266A/290R, 127L/134T, 127L/134T/160G/172R/210R/266A/290R, 127L/134T/192S/210R, 127L/134T/198Q/210R/290R, 127L/134T/210R/333T, 134R, 134R/160G/172R/333T, 134R/172R/192S/290R/333T, 134R/192S/198Q, 134R/198Q, 134R/198Q/266A/333T, 134R/333T, 134T/160G/198Q/266A/290R, 134T/210R, and 134T/210R/290R.
In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5412 and one or more residue differences as compared to SEQ ID NO: 5412, selected from Q73K/I127L/P134R/A160G/I172R/R198Q/K290R, Q73K/I127L/P134R/A160G/R198Q/L333T, Q73K/I127L/P134R/I172R/A192S/V266A/L333T, Q73K/I127L/P134R/I172R/R198Q, Q73K/I127L/P134R/L333T, Q73K/I127L/P134T/A160G/H210R/V266A,
Q73K/I127L/P134T/H210R/K290R, Q73K/I127L/P134T/H210R/L333T, Q73K/I127L/P134T/K290R/L333T, Q73K/P134R, Q73K/P134R/A160G/I172R/K290R, Q73K/P134R/A160G/L333T, Q73K/P134R/I172R, Q73K/P134R/I172R/H210R/L333T, Q73K/P134R/I172R/V266A/L333T, Q73K/P134R/L333T, Q73K/P134R/R366H, Q73K/P134T/A160G/I172R/A192S/K290R,
Q73K/P134T/A160G/I172R/R198Q/L333T, Q73K/P134T/A160G/V266A, Q73K/P134T/R198Q/H21 OR, Q73K/P134T/R198Q/H210R/V266A, Q73K/P134T/H210R/L333T, 1127L/P134R, 1127L/P134R/A160G/I172R/A192S/L333T, 1127L/P134R/I172R/R198Q/V266A/L333T, 1127L/P134R/H210R/V266A/K290R, 1127L/P134T, 1127L/P134T/A160G/I172R/H210R/V266A/K290R, 1127L/P134T/A192S/H21 OR, I127L/P134T/R198Q/H210R/K290R, I127L/P134T/H210R/L333T, P134R, P134R/A160G/I172R/L333T, P134R/I172R/A192S/K290R/L333T, P134R/A192S/R198Q, P134R/R198Q, P134R/R198Q/V266A/L333T, P134R/L333T, P134T/A160G/R198Q/V266A/K290R, P134T/H210R, and P134T/H210R/K290R.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5422 and one or more residue differences as compared to SEQ ID NO: 5422, selected from 13, 13/178/381 , 13/178/381/473, 13/178/457, 13/381 , 13/381/457, 13/402/457, 13/457, 178, 178/381/457, 178/381/457/473, 178/402/457, 178/473, 381 , 381/393/457, 381/457/473, 381/473, and 393/457. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5422 and one or more residue differences as compared to SEQ ID NO: 5422, selected from 13L, 13L/178R/381 D/473G, 13L/178R/381W, 13L/178R/457M, 13L/381 D/457M, 13L/381W, 13L/402L/457M, 13L/457M, 178R, 178R/381 D/457M/473G, 178R/381 W/457M, 178R/402L/457M, 178R/473G, 381 D/457M/473G, 381 W, 381 W/393T/457M, 381 W/473G, and 393T/457M. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5422 and one or more residue differences as compared to SEQ ID NO: 5422, selected from H13L, H13L/S178R/V381 D/E473G, H13L/S178R/V381 W, H13L/S178R/S457M, H13L/V381 D/S457M, H13L/V381W, H13L/K402L/S457M, H13L/S457M, S178R,
S178R/V381 D/S457M/E473G, S178R/V381 W/S457M, S178R/K402L/S457M, S178R/E473G, V381 D/S457M/E473G, V381W, V381 W/A393T/S457M, V381W/E473G, and A393T/S457M.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from 38/73/113/115/117/118/127/134/152/154/160/163/164/168/169/172/184/192/194/198/220/243/260/266/27 0/295/317/333/392/423, 38/73/113/115/117/118/127/134/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/29 5/317/333/392/423, 38/73/113/115/117/118/127/134/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/29 5/317/333/392/423/457, 38/73/113/115/117/118/127/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/295/31 7/333/392/423, 38/73/1 13/1 15/1 18/127/152/154/160/162/164/168/172/184/192/198/220/243/260/266/270/295/317/333/42 3,
38/73/1 13/1 15/1 18/127/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/295/317/33 3/423,
38/73/1 13/1 15/1 18/127/152/154/160/164/168/172/184/189/192/198/220/243/260/266/270/295/317/333/42
3,
38/73/1 13/1 15/1 18/127/152/154/160/164/168/172/184/192/198/210/220/243/260/266/270/295/317/333/42 3, 38/73/1 13/1 15/1 18/127/152/154/160/164/168/172/184/192/198/220/243/260/266/270/295/317/333/423, 38/73/1 13/1 18/127/152/154/160/164/168/172/184/192/198/220/243/260/266/270/295/317/333/423, 38/73/1 13/1 18/127/152/154/160/164/168/172/184/192/198/220/243/266/270/317/333/423,
38/73/1 13/1 18/127/152/154/160/164/168/172/184/192/198/220/266/270/317/333/423, and
38/1 13/127/152/154/160/164/168/172/198/220/290/317/423. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from
38F/73Q/1 13E/1 15E/1 17R/118R/1271/134R/152T/154K/160A/163V/164R/168K/169T/1721/184A/192A/19 8R/220R/243G/260I/266V/270P/295I/317M/333L/392A/423D,
38F/73Q/1 13E/1 15E/1 17R/1 18R/1271/134R/152T/154K/160A/163V/164R/168K/169T/1721/184A/192A/19 8R/220R/243G/260I/266V/270P/295I/317M/333L/392A/423D/457M,
38F/73Q/1 13E/1 15E/1 17R/1 18R/1271/134V/152T/154K/160A/163V/164R/168K/169T/1721/184A/192A/194 M/198R/220R/243G/260I/266V/270P/295I/317M/333L/392A/423D,
38F/73Q/1 13E/1 15E/1 17R/1 18R/1271/152T/154K/160A/163V/164R/168K/169T/1721/184A/192A/198R/22 0R/243G/260I/266V/270P/295I/317M/333L/392A/423D,
38F/73Q/1 13E/1 15E/1 18S/1271/152T/154K/160A/163V/164R/168M/169V/1721/184A/192A/198R/220R/24 3G/260I/266V/270P/295I/317M/333L/423D,
38F/73Q/1 13E/1 15Q/1 18S/1271/152T/154K/160A/162L/164R/168K/1721/184A/192A/198R/220R/243G/26 0I/266V/270P/295I/317M/333L/423D,
38F/73Q/1 13E/1 15Q/1 18S/1271/152T/154K/160A/163V/164R/168M/169V/1721/184A/192A/198R/220R/24 3G/260I/266V/270P/295I/317M/333L/423D,
38F/73Q/1 13E/1 15Q/1 18S/1271/152T/154K/160A/164R/168K/1721/184A/189S/192A/198R/220R/243G/26 0I/266V/270P/295I/317M/333L/423D,
38F/73Q/1 13E/1 15Q/1 18S/1271/152T/154K/160A/164R/168K/1721/184A/192A/198R/210R/220R/243G/26 0I/266V/270P/295I/317M/333L/423D,
38F/73Q/1 13E/1 15Q/1 18S/1271/152T/154K/160A/164R/168K/1721/184A/192A/198R/220R/243G/260I/266 V/270 P/2951/317M/333L/423D,
38F/73Q/1 13E/1 18S/1271/152T/154K/160A/164R/168K/1721/184A/192A/198R/220R/243G/260I/266V/270 P/2951/317M/333L/423D,
38F/73Q/1 13E/1 18S/1271/152T/154K/160A/164R/168K/1721/184A/192A/198 R/220R/243G/266 V/270 P/31 7M/333L/423D, 38F/73Q/1 13E/1 18S/1271/152T/154K/160A/164R/168K/1721/184A/192A/198R/220R/266V/270P/317M/33 3L/423D, and 38F/1 13E/1271/152T/154K/160A/164R/168K/1721/198R/220R/290R/317M/423D. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 972 and one or more residue differences as compared to SEQ ID NO: 972, selected from
C38F/K73Q/S1 13E/K1 15E/K1 17R/A1 18R/L127I/T134R/E152T/A154K/G160A/A163V/D164R/F168K/C16 9T/R172I/C184A/S192A/Q198R/P220R/V243G/L260I/A266V/K270P/L295I/R317M/T333L/C392A/W423D, C38F/K73Q/S1 13E/K1 15E/K1 17R/A1 18R/L127I/T134R/E152T/A154K/G160A/A163V/D164R/F168K/C16 9T/R172I/C184A/S192A/Q198R/P220R/V243G/L260I/A266V/K270P/L295I/R317M/T333L/C392A/W423D/ S457M,
C38F/K73Q/S1 13E/K1 15E/K1 17R/A1 18R/L127I/T134V/E152T/A154K/G160A/A163V/D164R/F168K/C16 9T/R172I/C184A/S192A/L194M/Q198R/P220R/V243G/L260I/A266V/K270P/L295I/R317M/T333L/C392A/ W423D,
C38F/K73Q/S1 13E/K1 15E/K1 17R/A1 18R/L127I/E152T/A154K/G160A/A163V/D164R/F168K/C169T/R17 2I/C184A/S192A/Q198R/P220R/V243G/L260I/A266V/K270P/L295I/R317M/T333L/C392A/W423D, C38F/K73Q/S1 13E/K1 15E/A1 18S/L127I/E152T/A154K/G160A/A163V/D164R/F168M/C169V/R172I/C184 A/S192A/Q198R/P220R/V243G/L260I/A266V/K270P/L295I/R317M/T333L/W423D,
C38F/K73Q/S1 13E/K1 15Q/A1 18S/L127I/E152T/A154K/G160A/W162L/D164R/F168K/R172I/C184A/S192 A/Q198R/P220R/V243G/L260I/A266V/K270P/L295I/R317M/T333L/W423D,
C38F/K73Q/S1 13E/K1 15Q/A1 18S/L127I/E152T/A154K/G160A/A163V/D164R/F168M/C169V/R172I/C184 A/S192A/Q198R/P220R/V243G/L260I/A266V/K270P/L295I/R317M/T333L/W423D,
C38F/K73Q/S1 13E/K1 15Q/A1 18S/L127I/E152T/A154K/G160A/D164R/F168K/R172I/C184A/D189S/S192 A/Q198R/P220R/V243G/L260I/A266V/K270P/L295I/R317M/T333L/W423D,
C38F/K73Q/S1 13E/K1 15Q/A1 18S/L127I/E152T/A154K/G160A/D164R/F168K/R172I/C184A/S192A/Q198 R/H210R/P220R/V243G/L260I/A266V/K270P/L295I/R317M/T333L/W423D,
C38F/K73Q/S1 13E/K1 15Q/A1 18S/L127I/E152T/A154K/G160A/D164R/F168K/R172I/C184A/S192A/Q198 R/P220 R/V243G/L2601/A266 V/K270 P/L2951/R317M/T333L/W423 D ,
C38F/K73Q/S1 13E/A1 18S/L127I/E152T/A154K/G160A/D164R/F168K/R172I/C184A/S192A/Q198R/P220 R/V243G/L2601/A266 V/K270 P/L2951/R317M/T333L/W423 D ,
C38F/K73Q/S1 13E/A1 18S/L127I/E152T/A154K/G160A/D164R/F168K/R172I/C184A/S192A/Q198R/P220 R/V243G/A266V/K270P/R317M/T333L/W423D,
C38F/K73Q/S1 13E/A1 18S/L127I/E152T/A154K/G160A/D164R/F168K/R172I/C184A/S192A/Q198R/P220 R/A266V/K270P/R317M/T333L/W423D, and
C38F/S1 13E/L127I/E152T/A154K/G160A/D164R/F168K/R172I/Q198R/P220R/K290R/R317M/W423D.
In some embodiments, the engineered transposase polypeptide with one or more improved properties comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5704 and one or more residue differences as compared to SEQ ID NO: 5704, selected from 48, 1 16, 1 18, 120, 130, 137, 153, 157, 163, 175, 181 , 187, 192, 213, 246, 263, 273, 296, 318, 334, 341 , 394, 412, 424, 454, and 458. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5704 and one or more residue differences as compared to SEQ ID NO: 5704, selected from 48V, 116E, 118E, 120R, 1301, 137R, 153H, 157K, 163A, 1751, 181 R, 187A, 192S, 213R, 246G, 263I, 273P, 296I, 318M, 334L, 341 L, 394A, 412N, 424D, 454R, and 458M. In some embodiments, the engineered transposase polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 5704 and one or more residue differences as compared to SEQ ID NO: 5706, selected from I48V, S116E, Q118E, K120R, L130I, F137R, D153H, A157K, G163A, R175I, S181 R, C187A, D192S, H213R, V246G, L263I, K273P, L296I, 1318M, T334L, Q341 L, E394A, K412N, W424D, E454R, and S458M.
As will be appreciated by the skilled artisan, in some embodiments, one or a combination of residue differences above that is selected can be kept constant (i.e., maintained) in the engineered transposase as a core feature, and additional residue differences at other residue positions incorporated into the sequence to generate additional engineered transposase polypeptides with improved properties. Accordingly, it is to be understood for any engineered transposase containing one or a subset of the residue differences above, the present invention contemplates other engineered transposases that comprise the one or subset of the residue differences, and additionally one or more residue differences at the other residue positions disclosed herein.
As noted above, the engineered transposase polypeptides are also capable of converting substrates (e.g., an adapter or donor polynucleotide and a target polynucleotide) to products (e.g., an annealed or ligated polynucleotide consisting of the adapter and the fragmented target polynucleotide). In some embodiments, the engineered transposase polypeptide is capable of converting the substrate compounds to the product compound with at least 1 .2 fold, 1 .5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, or more activity relative to the activity of the reference polypeptide of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704.
In some embodiments, the engineered transposase capable of converting the substrate compounds to the product compounds with at least 2-fold the activity relative to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, and comprises an amino acid sequence selected from the polypeptide sequences in SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756.
In some embodiments, the engineered transposase has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, that increases soluble expression or isolated protein yield of the engineered transposase in a bacterial host cell, particularly in E. coli, as compared to a wild-type or engineered reference transposase, and comprises an amino acid sequence selected from the polypeptide sequences in SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756.
In some embodiments, the engineered transposase has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, that increases thermostability of the engineered transposase, as compared to a wild-type or engineered reference transposase, and comprises an amino acid sequence selected from the polypeptide sequences in SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756.
In some embodiments, the engineered transposase has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, that increases the activity of the engineered transposase at high temperatures (by way of example and not limitation, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, or 65 °C), as compared to a wild-type or engineered reference transposase, and comprises an amino acid sequence selected from the polypeptide sequences in SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756.
In some embodiments, the engineered transposase has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, that reduces the sequence insertion bias of the engineered transposase, as compared to a wild-type or engineered reference transposase, and comprises an amino acid sequence selected from the polypeptide sequences in SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756.
In some embodiments, the engineered transposase has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, that reduces the inhibition, as compared to a wild-type or engineered reference transposase, and comprises an amino acid sequence selected from the polypeptide sequences in SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756.
In some embodiments, the engineered transposase has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, that increases specific activity of the engineered transposase on one or more an adapter or donor polynucleotide and target polynucleotide substrates as compared to a wild-type or engineered reference transposase, and comprises an amino acid sequence selected from the polypeptide sequences in SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756.
In some embodiments, the engineered transposase with one or more improved properties has an amino acid sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from the polypeptide sequences of SEQ ID NOs: 2-2368, 2388-5694, and 5706-5756. In some embodiments, the engineered transposase with one or more improved properties has an amino acid sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704.
In some embodiments, the engineered transposase with one or more improved properties has an amino acid sequence comprising a sequence selected from the polypeptide sequences of SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756. In some embodiments, the engineered transposase with one or more improved properties has an amino acid sequence comprising a sequence selected from SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704.
In some embodiments, the engineered transposase, comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to one of the polypeptide sequences of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, as provided in the Examples.
In some embodiments, exemplary engineered transposases useful in the methods described herein comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13) polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to, comprising the amino acid sequences of, or consisting the amino acid sequences of SEQ ID NOs: 4, 10, 396, 400, 608, 2000, 2002, 2014, 2058, 4000, 4012, 4560, 5000, and 5406.
In some embodiments, exemplary engineered transposases useful in the methods described herein comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13) polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to, comprising the amino acid sequences of, or consisting the amino acid sequences of SEQ ID NOs: 8, 40, 308, 900, 1058, 1208, 1688, 2246, 3460, 3490, 3982, 4864, 5026, and 5378.
In some embodiments, exemplary engineered transposases useful in the methods described herein comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13) polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to, comprising the amino acid sequences of, or consisting the amino acid sequences of SEQ ID NOs: 78, 146, 342, 746, 862, 894, 1006, 1668, 2444, 3680, 4782, 5234, 5436, and 5686.
In some embodiments, exemplary engineered transposases useful in the methods described herein comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13) polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to, comprising the amino acid sequences of, or consisting the amino acid sequences of SEQ ID NOs: 2, 972, 2368, 2980, 4144, 4642, 4934, 5196, 5286, 5300, 5500, 5656, 5692, and 5702.
In some embodiments, exemplary engineered transposases useful in the methods described herein comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13) polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to, comprising the amino acid sequences of, or consisting the amino acid sequences of SEQ ID NOs: 14, 68, 80, 92, 100, 506, 1004, 2090, 3456, 4562, 4674, 4682, 4790, 5002, and 5604.
In some embodiments, exemplary engineered transposases useful in the methods described herein comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13) polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to, comprising the amino acid sequences of, or consisting the amino acid sequences of SEQ ID NOs: 58, 780, 1008, 1010, 1218, 1788, 2680, 2712, 2686, 3648, 4814, 5034, 5270, 5578, and 5688.
In some embodiments, exemplary engineered transposases useful in the methods described herein comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13) polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to, comprising the amino acid sequences of, or consisting the amino acid sequences of SEQ ID NOs: 4, 90, 98, 302, 1000, 1446, 1488, 1806, 2574, 3285, 4672, 4922, 5066, 5646, and 5694.
In some embodiments, exemplary engineered transposases useful in the methods described herein comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13) polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to, comprising the amino acid sequences of, or consisting the amino acid sequences of SEQ ID NOs: 14, 974, 1162, 1782, 2422, 2682, 4680, 4886, 4924, 5132, 5346, 5422, 5588, 5624, and 5680.
In some embodiments, exemplary engineered transposases useful in the methods described herein comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13) polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to, comprising the amino acid sequences of, or consisting the amino acid sequences of SEQ ID NOs: 5706-5756. In some embodiments, exemplary engineered transposases useful in the methods described herein comprise one or more polypeptide sequences selected from polypeptide sequences comprising at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identities to the amino acid sequences of SEQ ID NOs: 5706-5756, wherein the polypeptide sequence has at least one mutation selected from the group consisting of I48V, S116E, Q118E, K120R, L130I, F137R, D153H, A157K, G163A, R175I, S181 R, C187A, D192S, H213R, V246G, L263I, K273P, L296I, 1318M, T334L, Q341 L, E394A, K412N, W424D, E454R, and S458M. In addition to the residue positions specified above, any of the engineered transposase polypeptides disclosed herein can further comprise other residue differences relative to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, at other residue positions (i.e., residue positions other than those included herein). Residue differences at these other residue positions can provide for additional variations in the amino acid sequence without adversely affecting the ability of the polypeptide to carry out the conversion of substrate to product. Accordingly, in some embodiments, in addition to the amino acid residue differences present in any one of the engineered transposase polypeptides selected from the polypeptide sequences in the range of SEQ ID NOs: 2-2368, 2388-5694, and 5706-5756, the sequence can further comprise 1 -2, 1 -3, 1 -4, 1 -5, 1 -6, 1 -7, 1 -8, 1 -9, 1 -10, 1 -1 1 , 1 -12, 1 -14, 1 -15, 1 -16, 1 -18, 1 -20, 1 -22, 1 -24, 1 - 26, 1 -30, 1 -35, 1 -40, 1 -45, 1 -50, 1 -100, or 1 -150 residue differences at other amino acid residue positions as compared to the SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704. In some embodiments, the number of amino acid residue differences as compared to the reference sequence can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 30, 35, 40, 45, 50, 100, or 150 residue positions. In some embodiments, the number of amino acid residue differences as compared to the reference sequence can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 18, 20, 21 , 22, 23, 24, or 25 residue positions. The residue differences at these other positions can be conservative changes or non-conservative changes. In some embodiments, the residue differences can comprise conservative substitutions and non-conservative substitutions as compared to the transposase polypeptide of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704.
In some embodiments, the present invention also provides engineered polypeptides that comprise a fragment of any of the engineered transposase polypeptides described herein that retains the functional activity and/or improved property of that engineered transposase. Accordingly, in some embodiments, the present invention provides a polypeptide fragment capable of converting substrate to product under suitable reaction conditions, wherein the fragment comprises at least about 90%, 95%, 96%, 97%, 98%, or 99% of a full-length or truncated amino acid sequence of an engineered transposase of the present invention, such as an exemplary transposase polypeptide selected from the polypeptide sequences in the range of SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756. In some embodiments, the engineered transposase can have an amino acid sequence comprising a deletion in any one of the transposase polypeptide sequences described herein, such as the exemplary engineered polypeptides of the polypeptide sequences in the range of SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756.
Thus, for each and every embodiment of the engineered transposase polypeptides of the invention, the amino acid sequence can comprise deletions of one or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids of the transposase polypeptides, where the associated functional activity and/or improved properties of the engineered transposase described herein are maintained. In some embodiments, the deletions can comprise 1 -2, 1 -3, 1 -4, 1 -5, 1 -6, 1 -7, 1 -8, 1 -9, 1 -10, 1 -15, 1 -20, 1 -21 , 1 - 22, 1 -23, 1 -24, 1 -25, 1 -30, 1 -35, 1 -40, 1 -45, or 1 -50 amino acid residues. In some embodiments, the number of deletions can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residues. In some embodiments, the deletions can comprise deletions of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 18, 20, 21 , 22, 23, 24, or 25 amino acid residues. In some embodiments, the engineered transposase polypeptide herein can have an amino acid sequence comprising an insertion as compared to any one of the engineered transposase polypeptides described herein, such as the exemplary engineered polypeptides of the polypeptide sequences in the range of SEQ ID NOs: 2-2368, 2388-5694, and 5706-5756. Thus, for each and every embodiment of the transposase polypeptides of the invention, the insertions can comprise one or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, or 50 or more amino acids, where the associated functional activity and/or improved properties of the engineered transposase described herein is maintained. The insertions can be to amino or carboxy terminus, or internal portions of the transposase polypeptide.
In some embodiments, the engineered transposase described herein can have an amino acid sequence comprising a sequence selected from the polypeptide sequences in the range of SEQ ID NOs: 4-2368, 2388-5694, and 5706-5756, and optionally one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1 -2, 1 -3, 1 -4, 1 -5, 1 -6, 1 -7, 1 -8, 1 -9, 1 -10, 1 -15, 1 -20, 1 -21 , 1 -22, 1 -23, 1 -24, 1 -25, 1 -30, 1 - 35, 1 -40, 1 -45, 1 -50, 1 -75, 1 -100, or 1 -150 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally around 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 1 10, 120, 130, 140, or 150 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the substitutions can be conservative or non-conservative substitutions.
In the above embodiments, the suitable reaction conditions for the engineered polypeptides are provided in Tables 5.1 , 6.1 , 7.1 , 8.1 , 9.1 , 10.1 , 10.4, 1 1 .1 , 1 1 .3, 12.1 , 12.4, 13.1 , 14.1 , 14.4, 14.7, 15.1 ,
16.2, 16.3, 17.2, 17.3, 18.2, 19.2, 19.3, 20.2, 20.3, 21 .2, 21 .3, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2,
29.2, 30.2, 31 .2, 32.2, 33.2, 34.2, 35.1 , 36.1 , and 36.3, and as described in the Examples herein.
In some embodiments, the polypeptides of the present invention are fusion polypeptides in which the engineered polypeptides are fused to other polypeptides, such as, by way of example and not limitation, antibody tags (e.g., myc epitope), purification sequences (e.g., His tags for binding to metals), and cell localization signals (e.g., secretion signals). Thus, the engineered polypeptides described herein can be used with or without fusions to other polypeptides.
It is to be understood that the polypeptides described herein are not restricted to the genetically encoded amino acids. In addition to the genetically encoded amino acids, the polypeptides described herein may be comprised, either in whole or in part, of naturally occurring and/or synthetic non-encoded amino acids. Certain commonly encountered non-encoded amino acids of which the polypeptides described herein may be comprised include, but are not limited to: the D-stereoisomers of the genetically- encoded amino acids; 2,3-diaminopropionic acid (Dpr); a-aminoisobutyric acid (Aib); e-aminohexanoic acid (Aha); 5-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine (Ocf); 3- chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf); 2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff); 4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf); 3-bromophenylalanine (Mbf); 4- bromophenylalanine (Pbf); 2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf); 4- methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf); 3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf); 2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf); 4-cyanophenylalanine (Pcf); 2- trifluoromethylphenylalanine (Otf); 3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine (Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif); 4-aminomethylphenylalanine (Pamf); 2,4- dichlorophenylalanine (Opef); 3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff); 3,4- difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla); pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1 -ylalanine (1 nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla); benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla); homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp); pentafluorophenylalanine (5ff) ; styrylkalanine (sAla); authrylalanine (aAla); 3,3- diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp); penicillamine (Pen); 1 ,2,3,4- tetrahydroisoquinoline-3-carboxylic acid (Tic); p-2-thienylalanine (Thi); methionine sulfoxide (Mso); N(w)- nitroarginine (nArg); homolysine (hLys); phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer); phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid (hGlu); 1 -aminocyclopent-(2 or 3)- ene-4 carboxylic acid; pipecolic acid (PA), azetidine-3-carboxylic acid (ACA); 1 -aminocyclopentane-3- carboxylic acid; allylglycine (aGly); propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal); homoleucine (hLeu), homovaline (hVal); homoisoleucine (hlle); homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal); homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) and homoproline (hPro). Additional nonencoded amino acids of which the polypeptides described herein may be comprised will be apparent to those of skill in the art (See e.g., the various amino acids provided in Fasman, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Boca Raton, FL, pp. 3-70 [1989], and the references cited therein, all of which are incorporated by reference). These amino acids may be in either the L- or □-configuration.
Those of skill in the art will recognize that amino acids or residues bearing side chain protecting groups may also comprise the polypeptides described herein. Non-limiting examples of such protected amino acids, which in this case belong to the aromatic category, include (protecting groups listed in parentheses), but are not limited to: Arg(tos), Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(8- benzylester), Gln(xanthyl), Asn(N-S-xanthyl), His(bom), His(benzyl), His(tos), Lys(fmoc), Lys(tos), Ser(O- benzyl), Thr (O-benzyl) and Tyr(O-benzyl).
Non-encoding amino acids that are conformationally constrained of which the polypeptides described herein may be composed include, but are not limited to, N-methyl amino acids (L-configuration); 1 -aminocyclopent-(2 or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylic acid; homoproline (hPro); and 1 -aminocyclopentane-3-carboxylic acid.
In some embodiments, the engineered polypeptides can be in various forms, for example, such as an isolated preparation, as a substantially purified enzyme, whole cells transformed with gene(s) encoding the enzyme, and/or as cell extracts and/or lysates of such cells. The enzymes can be lyophilized, spray-dried, precipitated or be in the form of a crude paste, as further discussed below. In some embodiments, the engineered polypeptides can be in the form of a biocatalytic composition. In some embodiments, the biocatalytic composition comprises (a) a means for conversion of an adapter or donor polynucleotide and a target polynucleotide to an annealed or ligated polynucleotide consisting of the adapter and the optionally fragmented target polynucleotide by contact with a transposase and (b) a suitable cofactor. The suitable cofactor may be magnesium, manganese, or any other suitable cofactor.
In some embodiments, the polypeptides described herein are provided in the form of kits. The enzymes in the kits may be present individually or as a plurality of enzymes. The kits can further include reagents for carrying out the enzymatic reactions, substrates for assessing the activity of enzymes, as well as reagents for detecting the products. The kits can also include reagent dispensers and instructions for use of the kits.
In some embodiments, the kits of the present invention include arrays comprising a plurality of different transposase polypeptides at different addressable position, wherein the different polypeptides are different variants of a reference sequence each having at least one different improved enzyme property. In some embodiments, a plurality of polypeptides immobilized on solid supports are configured on an array at various locations, addressable for robotic delivery of reagents, or by detection methods and/or instruments. The array can be used to test a variety of substrate compounds for conversion by the polypeptides. Such arrays comprising a plurality of engineered polypeptides and methods of their use are known in the art (See e.g., W02009/008908A2).
Polynucleotides Encoding Engineered Transposases, Expression Vectors and Host Cells
In another aspect, the present invention provides polynucleotides encoding the engineered transposase polypeptides described herein. The polynucleotides may be operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. Expression constructs containing a heterologous polynucleotide encoding the engineered transposase are introduced into appropriate host cells to express the corresponding transposase polypeptide.
As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremely large number of nucleic acids to be made, all of which encode the improved transposase enzymes. Thus, having knowledge of a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the protein. In this regard, the present invention specifically contemplates each and every possible variation of polynucleotides that could be made encoding the polypeptides described herein by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 10.3, 10.5, 11 .2, 11 .4, 11 .5, 12.2, 12.3, 12.5, 12.6, 13.2, 13.3, 13.4, 14.2, 14. 3, 14.5, 14.6, 14.8, 14.9, 15.1 , 16.2, 16.3, 17.2, 17.3, 18.2, 19.2, 19.3, 20.2, 20.3, 21 .2, 21 .3, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31 .2, 32.2,
33.2, 34.2, 35.1 , 36.1 , and 36.3, and disclosed in the sequence listing incorporated by reference herein as the polypeptide sequences in the range of SEQ ID NOs: 2-2368, 2388-5694, and 5706-5756.
In various embodiments, the codons are preferably selected to fit the host cell in which the protein is being produced. For example, preferred codons used in bacteria are used to express the gene in bacteria; preferred codons used in yeast are used for expression in yeast; and preferred codons used in mammals are used for expression in mammalian cells. In some embodiments, all codons need not be replaced to optimize the codon usage of the transposase since the natural sequence will comprise preferred codons and because use of preferred codons may not be required for all amino acid residues. Consequently, codon optimized polynucleotides encoding the transposase enzymes may contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of the full- length coding region.
In some embodiments, the polynucleotide comprises a codon optimized nucleotide sequence encoding the transposase polypeptide amino acid sequence, as represented by SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704. In some embodiments, the polynucleotide has a nucleic acid sequence comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the codon optimized nucleic acid sequences encoding the polypeptide sequences in the range of SEQ ID NOs: 2-2368, 2388-5694, and 5706-5756. In some embodiments, the polynucleotide has a nucleic acid sequence comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the codon optimized nucleic acid sequences in the polynucleotide sequences in the range of SEQ ID NOs: 1 -2367, 2387-5693, and 5705-5755. In some embodiments, the codon optimized sequences of the polynucleotide sequences in the range of SEQ ID NOs: 1 -2367, 2387-5693, and 5705-5755, enhance expression of the encoded transposase, providing preparations of enzyme capable of converting substrate to product.
In some embodiments, the polynucleotide sequence comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NOs: 1 , 3, 155, 301 , 337, 605, 971 , 1081 , 1217, 1579, 1597, 1599, 1603, 1719, 2367, 2425, 2539, 3401 , 3661 , 3787, 4149, 4277, 4641 , 4983, 5195, 5285, 5411 , 5421 , and/or 5703, and/or or a functional fragment thereof, wherein said polynucleotide sequence encodes an engineered polypeptide comprising at least one substitution at one or more amino acid positions.
In some embodiments, the polynucleotide sequence encodes at least one engineered transposase comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704.
In some embodiments, the polynucleotide sequence comprises SEQ ID NOs: 1 , 3, 155, 301 , 337, 605, 971 , 1081 , 1217, 1579, 1597, 1599, 1603, 1719, 2367, 2425, 2539, 3401 , 3661 , 3787, 4149, 4277, 4641 , 4983, 5195, 5285, 5411 , 5421 , and/or 5703. In some embodiments, the polynucleotides are capable of hybridizing under highly stringent conditions to a reference sequence selected from the polynucleotide sequences in SEQ ID NOs: 1 -2367, 2387-5693, and 5705-5755, or a complement thereof, and encode a transposase.
In some embodiments, as described above, the polynucleotide encodes an engineered transposase polypeptide with improved properties as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, and one or more residue differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, wherein the sequence is selected from the polypeptide sequences in the range of SEQ ID NOs: 2-2368, 2388-5694, and 5706- 5756. In some embodiments, the reference amino acid sequence is selected from the polypeptide sequences in the range of SEQ ID NOs: 2-2368, 2388-5694, and 5704. In some embodiments, the reference amino acid sequence is SEQ ID NO: 2, while in some other embodiments, the reference sequence is SEQ ID NO: 4, while in some other embodiments, the reference sequence is SEQ ID NO: 156. In some embodiments, the reference amino acid sequence is SEQ ID NO: 302, while in some other embodiments, the reference sequence is SEQ ID NO: 338, while in some other embodiments, the reference sequence is SEQ ID NO: 606. In some embodiments, the reference amino acid sequence is SEQ ID NO: 972, while in some other embodiments, the reference sequence is SEQ ID NO: 1082, while in some other embodiments, the reference sequence is SEQ ID NO: 1218. In some embodiments, the reference amino acid sequence is SEQ ID NO: 1580, while in some other embodiments, the reference sequence is SEQ ID NO: 1598, while in some other embodiments, the reference sequence is SEQ ID NO: 1600. In some embodiments, the reference amino acid sequence is SEQ ID NO: 1604, while in some other embodiments, the reference sequence is SEQ ID NO: 1720, while in some other embodiments, the reference sequence is SEQ ID NO: 2368. In some embodiments, the reference amino acid sequence is SEQ ID NO: 2426, while in some other embodiments, the reference sequence is SEQ ID NO: 2540, while in some other embodiments, the reference sequence is SEQ ID NO: 3402. In some embodiments, the reference amino acid sequence is SEQ ID NO: 3662, while in some other embodiments, the reference sequence is SEQ ID NO: 3788, while in some other embodiments, the reference sequence is SEQ ID NO: 4150. In some embodiments, the reference amino acid sequence is SEQ ID NO: 4278, while in some other embodiments, the reference sequence is SEQ ID NO: 4642, while in some other embodiments, the reference sequence is SEQ ID NO: 4984. In some embodiments, the reference amino acid sequence is SEQ ID NO: 5196, while in some other embodiments, the reference sequence is SEQ ID NO: 5286, while in some other embodiments, the reference sequence is SEQ ID NO: 5412. In some embodiments, the reference amino acid sequence is SEQ ID NO: 5422. In some embodiments, the reference amino acid sequence is SEQ ID NO: 5500. In some embodiments, the reference amino acid sequence is SEQ ID NO: 5704. In some embodiments, the polynucleotide encodes a transposase polypeptide capable of converting one or more substrates to product with improved properties as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704.
In some embodiments, the polynucleotide encoding the engineered transposase comprises a polynucleotide sequence selected from the polynucleotide sequences in the range of SEQ ID NOs: 1 - 2367, 2387-5693, and 5705-5755.
In some embodiments, the polynucleotides are capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from the polynucleotide sequences in the range of SEQ ID NOs: 1 -2367, 2387-5693, and 5705-5755 or a complement thereof, and encode a transposase polypeptide with one or more of the improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a transposase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, that has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, as described above and in the Examples, below.
In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered transposase polypeptide with improved properties comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704. In some embodiments, the polynucleotides encode the polypeptides described herein but have at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding the engineered transposase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 1 -2367, 2387-5693, and 5705-5755.
In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered transposase polypeptide with improved properties comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704. In some embodiments, the polynucleotides encode the polypeptides described herein but have at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding the engineered transposase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 1 -2367, 2387-5693, and 5705-5755.
In some embodiments, an isolated polynucleotide encoding any of the engineered transposase polypeptides provided herein is manipulated in a variety of ways to provide for expression of the polypeptide. In some embodiments, the polynucleotides encoding the polypeptides are provided as expression vectors where one or more control sequences is present to regulate the expression of the polynucleotides and/or polypeptides. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.
In some embodiments, the control sequences include among other sequences, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. As known in the art, suitable promoters can be selected based on the host cells used. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present application, include, but are not limited to the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (See e.g., Villa- Kamaroff et al., Proc. Natl Acad. Sci. USA 75: 3727-3731 [1978]), as well as the tac promoter (See e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80: 21 -25 [1983]). Exemplary promoters for filamentous fungal host cells, include promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alphaamylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes can be from the genes for Saccharomyces cerevisiae enolase (ENO-1 ), Saccharomyces cerevisiae galactokinase (GAL1 ), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (See e.g., Romanos et al., Yeast 8:423-488 [1992]).
In some embodiments, the control sequence is a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice finds use in the present invention. For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1 ), and Saccharomyces cerevisiae glyceraldehyde-3- phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (See e.g., Romanos et al., supra).
In some embodiments, the control sequence is a suitable leader sequence, a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells include, but are not limited to those obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1 ), Saccharomyces cerevisiae 3- phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP). The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to those from the genes for Aspergillus oryzae AKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsinlike protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (See e.g., Guo and Sherman, Mol. Cell. Bio., 15:5983-5990 [1995]).
In some embodiments, the control sequence is a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. Any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered transposase polypeptides provided herein. Effective signal peptide coding regions for bacterial host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (See e.g., Simonen and Palva, Microbiol. Rev., 57:109-137 [1993]). Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae AKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. In some embodiments, the control sequence is a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is referred to as a “proenzyme,” “propolypeptide,” or “zymogen,” in some cases). A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region includes, but is not limited to the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (See e.g., WO 95/33836). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.
In some embodiments, regulatory sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to the ADH2 system or GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to the TAKA alphaamylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.
The present invention also provides recombinant expression vectors comprising a polynucleotide encoding an engineered transposase polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. In some embodiments, the various nucleic acid and control sequences described above are combined together to produce a recombinant expression vector which includes one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the variant transposase polypeptide at such sites. Alternatively, the polynucleotide sequence(s) of the present invention are expressed by inserting the polynucleotide sequence or a nucleic acid construct comprising the polynucleotide sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and can result in the expression of the variant transposase polynucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.
In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some alternative embodiments, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
In some embodiments, the expression vector preferably contains one or more selectable markers, which permit easy selection of transformed cells. A “selectable marker” is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophy, and the like. Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. In another aspect, the present invention provides a host cell comprising a polynucleotide encoding at least one engineered transposase polypeptide of the present invention, the polynucleotide being operatively linked to one or more control sequences for expression of the engineered transposase enzyme(s) in the host cell. Host cells for use in expressing the polypeptides encoded by the expression vectors of the present invention are well known in the art and include but are not limited to, bacterial cells, such as E. coll, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris [ATCC Accession No. 201178]); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells are Escherichia co// strains (e.g., W3110 (AfhuA) and BL21 ).
In some embodiments, the host cell strain comprises a knockout of one or more genes, in particular phosphatase genes. In some embodiments, the host cell comprises a knockout or single gene deletion of E. coll genes aphA, surE, phoA, and/or cpdB, as described below in the Examples. In some embodiments, the host cell comprising a knockout of one or more phosphatase genes has increased production of the product and/or decreased de-phosphorylation of the product or substrate.
Accordingly, in another aspect, the present invention provides methods for producing the engineered transposase polypeptides, where the methods comprise culturing a host cell capable of expressing a polynucleotide encoding the engineered transposase polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the methods further comprise the steps of isolating and/or purifying the transposase polypeptides, as described herein.
Appropriate culture media and growth conditions for the above-described host cells are well known in the art. Polynucleotides for expression of the transposase polypeptides may be introduced into cells by various methods known in the art. Techniques include, among others, electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion.
The engineered transposases with the properties disclosed herein can be obtained by subjecting the polynucleotide encoding the naturally occurring or engineered transposase polypeptide to mutagenesis and/or directed evolution methods known in the art, and as described herein. An exemplary directed evolution technique is mutagenesis and/or DNA shuffling (See e.g., Stemmer, Proc. Natl. Acad. Sci. USA 91 :10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651 ; WO 01/75767 and U.S. Pat. 6,537,746). Other directed evolution procedures that can be used include, among others, staggered extension process (StEP), in vitro recombination (See e.g., Zhao et al., Nat. Biotechnol., 16:258-261 [1998]), mutagenic PCR (See e.g., Caldwell et al., PCR Methods Appl., 3:S136-S140 [1994]), and cassette mutagenesis (See e.g., Black et al., Proc. Natl. Acad. Sci. USA 93:3525-3529 [1996]).
For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides to generate variant libraries that can be expressed, screened, and assayed. Mutagenesis and directed evolution methods are well known in the art (See e.g., US Patent Nos. 5,605,793, 5,811 ,238, 5,830,721 , 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251 ,674, 6,265,201 , 6,277,638, 6,287,861 , 6,287,862, 6,291 ,242, 6,297,053, 6,303,344, 6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861 , 6,372,497, 6,337,186, 6,376,246, 6,379,964, 6,387,702, 6,391 ,552, 6,391 ,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011 , 6,484,105, 6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603, 6,518,065, 6,519,065, 6,521 ,453, 6,528,311 , 6,537,746, 6,573,098, 6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072, 6,686,515, 6,703,240, 6,716,631 , 6,825,001 , 6,902,922, 6,917,882, 6,946,296, 6,961 ,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297, 7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421 ,347, 7,430,477, 7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464, 7,747,391 , 7,747,393, 7,751 ,986, 7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499, 7,904,249, 7,957,912, 7,981 ,614, 8,014,961 , 8,029,988, 8,048,674, 8,058,001 , 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681 , 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871 , 9,593,326, and all related US, as well as PCT and non-US counterparts; Ling et al., Anal. Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botstein et al., Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1 -7 [1986]; Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene, 34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284- 290 [1999]; Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameri et al., Nature, 391 :288-291 [1998]; Crameri, et al., Nat. Biotechnol., 15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319 [1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad. Sci. USA, 91 :10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651 ; WO 01/75767; and WO 2009/152336, all of which are incorporated herein by reference).
In some embodiments, the enzyme clones obtained following mutagenesis treatment are screened by subjecting the enzymes to a defined temperature (or other assay conditions, such as testing the enzyme’s activity over a broad range of substrates) and measuring the amount of enzyme activity remaining after heat treatments or other assay conditions. Clones containing a polynucleotide encoding a transposase polypeptide are then sequenced to identify the nucleotide sequence changes (if any), and used to express the enzyme in a host cell. Measuring enzyme activity from the expression libraries can be performed using any suitable method known in the art (e.g., standard biochemistry techniques, such as HPLC analysis).
In some embodiments, the clones obtained following mutagenesis treatment can be screened for engineered transposases having one or more desired improved enzyme properties (e.g., reduced sequence bias) Measuring enzyme activity from the expression libraries can be performed using the standard biochemistry techniques, such as HPLC analysis, LC-MS analysis, RapidFire-MS analysis, and/or capillary electrophoresis analysis.
When the sequence of the engineered polypeptide is known, the polynucleotides encoding the enzyme can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be individually synthesized, then joined {e.g., by enzymatic or chemical ligation methods, or polymerase mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides encoding portions of the transposase can be prepared by chemical synthesis as known in the art (e.g., the classical phosphoramidite method of Beaucage et al., Tet. Lett. 22:1859-69 [1981 ], or the method described by Matthes et al., EMBO J. 3:801 -05 [1984]) as typically practiced in automated synthetic methods. According to the phosphoramidite method, oligonucleotides are synthesized {e.g., in an automatic DNA synthesizer), purified, annealed, ligated and cloned in appropriate vectors. In addition, essentially any nucleic acid can be obtained from any of a variety of commercial sources. In some embodiments, additional variations can be created by synthesizing oligonucleotides containing deletions, insertions, and/or substitutions, and combining the oligonucleotides in various permutations to create engineered transposases with improved properties.
Accordingly, in some embodiments, a method for preparing the engineered transposase polypeptide comprises: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to an amino acid sequence selected from the polypeptide sequences of SEQ ID NOs: 2-2368 and 5704-5756, and having one or more residue differences as compared to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704; and (b) expressing the transposase polypeptide encoded by the polynucleotide.
In some embodiments of the method, the polynucleotide encodes an engineered transposase that has optionally one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1 -2, 1 -3, 1 -4, 1 -5, 1 - 6, 1 -7, 1 -8, 1 -9, 1 -10, 1 -15, 1 -20, 1 -21 , 1 -22, 1 -23, 1 -24, 1 -25, 1 -30, 1 -35, 1 -40, 1 -45, 1 -50, 1 -75, 1 -100, or 1 -150 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally around 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 1 10, 120, 130, 140, or 150 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the substitutions can be conservative or non-conservative substitutions. In some embodiments, any of the engineered transposase enzymes expressed in a host cell can be recovered from the cells and/or the culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography. Suitable solutions for lysing and the high efficiency extraction of proteins from bacteria, such as E. coli, are commercially available (e.g., CelLytic B™, Sigma- Aldrich, St. Louis MO).
Chromatographic techniques for isolation of the transposase polypeptide include, among others, reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art.
In some embodiments, affinity techniques may be used to isolate the improved transposase enzymes. For affinity chromatography purification, any antibody which specifically binds the transposase polypeptide may be used. For the production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., may be immunized by injection with a transposase polypeptide, or a fragment thereof. The transposase polypeptide or fragment may be attached to a suitable carrier, such as BSA, by means of a side chain functional group or linkers attached to a side chain functional group. In some embodiments, the affinity purification can use a specific ligand bound by the transposase or dye affinity column (See e.g., EP0641862; Stellwagen, “Dye Affinity Chromatography,” In Current Protocols in Protein Science, Unit 9.2-9.2.16 [2001]).
In some embodiments, a polynucleotide that encodes an engineered transposase (e.g., any one of the engineered transposases described herein, such as in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 10.3, 10.5, 11.2, 11.4, 11.5, 12.2, 12.3, 12.5, 12.6, 13.2, 13.3, 13.4, 14.2, 14. 3, 14.5, 14.6, 14.8, 14.9, 15.1 ,
16.2, 16.3, 17.2, 17.3, 18.2, 19.2, 19.3, 20.2, 20.3, 21 .2, 21 .3, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2,
29.2, 30.2, 31 .2, 32.2, 33.2, 34.2, 35.1 , 36.1 , and 36.3) is modified to replace the segment of nucleotides that encode the C-terminal 6-histidine tag with a different tag (e.g., a different affinity tag). In some embodiments, the polynucleotide that encodes an engineered transposase (e.g., any one of the engineered transposases described herein, such as in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 10.3, 10.5,
11.2, 11.4, 11.5, 12.2, 12.3, 12.5, 12.6, 13.2, 13.3, 13.4, 14.2, 14. 3, 14.5, 14.6, 14.8, 14.9, 15.1 , 16.2,
16.3, 17.2, 17.3, 18.2, 19.2, 19.3, 20.2, 20.3, 21 .2, 21 .3, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2,
30.2, 31 .2, 32.2, 33.2, 34.2, 35.1 , 36.1 , and 36.3) is modified to encode an engineered transposase with one or more additional tags (e.g., one or more affinity tags), such as the C-terminal 6-histidine tag and one or more additional tags. Tags such as affinity tags (e.g., affinity tags suitable for protein purification) are known in the art and include, for instance, a polyhistidine tag (e.g., five or more histidine residues), a polyarginine tag (e.g., five or more arginine residues), and a FLAG tag.
In some embodiments, a polynucleotide that encodes an engineered transposase (e.g., any one of the engineered transposases described herein, such as in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 10.3, 10.5, 11.2, 11.4, 11.5, 12.2, 12.3, 12.5, 12.6, 13.2, 13.3, 13.4, 14.2, 14. 3, 14.5, 14.6, 14.8, 14.9, 15.1 ,
16.2, 16.3, 17.2, 17.3, 18.2, 19.2, 19.3, 20.2, 20.3, 21 .2, 21 .3, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2,
29.2, 30.2, 31 .2, 32.2, 33.2, 34.2, 35.1 , 36.1 , and 36.3) is modified to replace the segment of nucleotides that encodes the C-terminal GGSG (SEQ ID NO: 5777) peptide spacer with a different peptide spacer. Suitable peptide spacers are known in the art and comprise flexible amino acid residues such as glycine, alanine, and serine.
In some embodiments, a peptide spacer includes motifs, such as multiple or repeating motifs, of GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 5778), GGGS (SEQ ID NO: 5779), GGGG (SEQ ID NO: 5780), GGGGA (SEQ ID NO: 5781 ), GGGGS (SEQ ID NO: 5782), GGGGG (SEQ ID NO: 5783), GGAG (SEQ ID NO: 5784), AGGG (SEQ ID NO: 5785), or SGGG (SEQ ID NO: 5786). In some embodiments, a peptide spacer includes 2 to 12 amino acids including motifs of GA or GS, e.g., GA, GS, GAGA (SEQ ID NO: 5787), GSGS (SEQ ID NO: 5788), GAGAGA (SEQ ID NO: 5789), GSGSGS (SEQ ID NO: 5790), GAGAGAGA (SEQ ID NO: 5791 ), GSGSGSGS (SEQ ID NO: 5792), GAGAGAGAGA (SEQ ID NO: 5793), GSGSGSGSGS (SEQ ID NO: 5794), GAGAGAGAGAGA (SEQ ID NO: 5795), and GSGSGSGSGSGS (SEQ ID NO: 5796).
In some embodiments, a peptide spacer includes 3 to 12 amino acids including motifs of GGA or GGS, e.g., GGA, GGS, GGAGGA (SEQ ID NO: 5797), GGSGGS (SEQ ID NO: 5798), GGAGGAGGA (SEQ ID NO: 5799), GGSGGSGGS (SEQ ID NO: 5780), GGAGGAGGAGGA (SEQ ID NO: 5801 ), and GGSGGSGGSGGS (SEQ ID NO: 5802).
In yet some embodiments, a peptide spacer includes 4 to 12 amino acids including motifs of GGAG (SEQ ID NO: 5803), GGAGGGAG (SEQ ID NO: 5804), GGSGGGSG (SEQ ID NO: 5805), GGAGGGAGGGAG (SEQ ID NO: 5806), and GGSGGGSGGGSG (SEQ ID NO: 5807). In some embodiments, a spacer includes motifs of GGGGA (SEQ ID NO: 5808), or GGGGS (SEQ ID NO: 5809), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 5810) and GGGGSGGGGSGGGGS (SEQ ID NO: 5811 ).
In some embodiments, a peptide spacer is cleavable. In some embodiments, a peptide spacer contains a protease binding site and can be cleaved by a protease via site-specific enzyme activity.
In some embodiments, an engineered transposase (e.g., an engineered transposase of the invention) is modified to include one or more tags and/or linkers on either the N- or C- termini. In some embodiments, a tag (e.g., a polyhistidine tag such as a 6-histidine tag) and/or a portion of a peptide spacer of an engineered transposase (e.g., an engineered transposase of the invention) is removed following or during purification. In some embodiments, the tag (e.g., an affinity tag) and/or a portion of the peptide spacer is removed, e.g., via proteolytic cleavage.
In some embodiments, a polynucleotide that encodes an engineered transposase (e.g., any one of the engineered transposases described herein, such as in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 10.3, 10.5, 11.2, 11.4, 11.5, 12.2, 12.3, 12.5, 12.6, 13.2, 13.3, 13.4, 14.2, 14. 3, 14.5, 14.6, 14.8, 14.9, 15.1 ,
16.2, 16.3, 17.2, 17.3, 18.2, 19.2, 19.3, 20.2, 20.3, 21 .2, 21 .3, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2,
29.2, 30.2, 31 .2, 32.2, 33.2, 34.2, 35.1 , 36.1 , and 36.3) is modified to remove the segment of nucleotides that encode the C-terminal 6-histidine tag and/or the C-terminal GGSG (SEQ ID NO: 5777) peptide spacer.
Methods of Using the Engineered Transposase Enzymes
In some embodiments, the transposase enzymes described herein find use in processes for conversion of one or more suitable substrates to a product. In some embodiments, the engineered transposase polypeptides disclosed herein can be used in a process for the conversion of an adapter or donor polynucleotide and a target polynucleotide to an annealed or ligated polynucleotide consisting of the adapter and the optionally fragmented target polynucleotide. In some embodiments, this process results in a pool of fragmented polynucleotides with ligated adapters, useful as a next generation sequencing library.
In the embodiments provided herein and illustrated in the Examples, various ranges of suitable reaction conditions that can be used in the processes, include but are not limited to, substrate loading, cosubstrate loading, pH, temperature, buffer, solvent system, cofactor, polypeptide loading, and reaction time. Further suitable reaction conditions for carrying out the process for biocatalytic conversion of substrate compounds to product compounds using an engineered transposase described herein can be readily optimized in view of the guidance provided herein by routine experimentation that includes, but is not limited to, contacting the engineered transposase polypeptide and one or more substrate compounds under experimental reaction conditions of concentration, pH, temperature, and solvent conditions, and detecting the product compound.
The substrate compound(s) in the reaction mixtures can be varied, taking into consideration, for example, the desired amount of product compound, the effect of each substrate concentration on enzyme activity, stability of enzyme under reaction conditions, and the percent conversion of each substrate to product. In some embodiments, the suitable reaction conditions comprise a substrate compound loading for each substrate of at least about 0.1 pM to 1 pM, 1 pM to 2 pM, 2 pM to 3 pM, 3 pM to 5 pM, 5 pM to 10 pM, or 10 pM or greater. In some embodiments, the suitable reaction conditions comprise a substrate compound loading for each substrate of at least about 0.5 to about 25 g/L, 1 to about 25 g/L, 5 to about 25 g/L, about 10 to about 25 g/L, or 20 to about 25 g/L. In some embodiments, the suitable reaction conditions comprise a substrate compound loading for each substrate of at least about 0.5 g/L, at least about 1 g/L, at least about 5 g/L, at least about 10 g/L, at least about 15 g/L, at least about 20 g/L, or at least about 30 g/L, or even greater.
In carrying out the transposase-mediated synthesis processes described herein, the engineered polypeptide may be added to the reaction mixture in the form of a purified enzyme, partially purified enzyme, whole cells transformed with gene(s) encoding the enzyme, as cell extracts and/or lysates of such cells, and/or as an enzyme immobilized on a solid support. Whole cells transformed with gene(s) encoding the engineered transposase enzyme or cell extracts, lysates thereof, and isolated enzymes may be employed in a variety of different forms, including solid (e.g., lyophilized, spray-dried, and the like) or semisolid (e.g., a crude paste). The cell extracts or cell lysates may be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment or the like, followed by a desalting procedure prior to lyophilization (e.g., ultrafiltration, dialysis, etc.). Any of the enzyme preparations (including whole cell preparations) may be stabilized by crosslinking using known crosslinking agents, such as, for example, glutaraldehyde or immobilization to a solid phase (e.g., Eupergit C, and the like).
The gene(s) encoding the engineered transposase polypeptides can be transformed into host cell separately or together into the same host cell. For example, in some embodiments one set of host cells can be transformed with gene(s) encoding one engineered transposase polypeptide, and another set can be transformed with gene(s) encoding another transposase. Both sets of transformed cells can be utilized together in the reaction mixture in the form of whole cells, or in the form of lysates or extracts derived therefrom. In other embodiments, a host cell can be transformed with gene(s) encoding multiple engineered transposase polypeptides. In some embodiments the engineered polypeptides can be expressed in the form of secreted polypeptides, and the culture medium containing the secreted polypeptides can be used for the transposase reaction.
In some embodiments, the improved activity of the engineered transposase polypeptides disclosed herein provides for processes wherein higher percentage conversion can be achieved with lower concentrations of the engineered polypeptide. In some embodiments of the process, the suitable reaction conditions comprise an engineered polypeptide amount of about 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 20% (w/w), 30% (w/w), 40% (w/w), 50% (w/w), 75% (w/w), 100% (w/w) or more of substrate compound loading.
In some embodiments, the engineered polypeptide is present at a molar ratio of engineered polypeptide to substrate of about 50 to 1 , 25 to 1 , 10 to 1 , 5 to 1 , 1 to 1 , 1 to 5, 1 to 10, 1 to 25 or 1 to 50. In some embodiments, the engineered polypeptide is present at a molar ratio of engineered polypeptide to substrate from a range of about 50 to 1 to a range of about 1 to 50.
In some embodiments, the engineered polypeptide is present at about 0.01 g/L to about 50 g/L; about 0.01 to about 0.1 g/L; about 0.05 g/L to about 50 g/L; about 0.1 g/L to about 40 g/L; about 1 g/L to about 40 g/L; about 2 g/L to about 40 g/L; about 5 g/L to about 40 g/L; about 5 g/L to about 30 g/L; about 0.1 g/L to about 10 g/L; about 0.5 g/L to about 10 g/L; about 1 g/L to about 10 g/L; about 0.1 g/L to about 5 g/L; about 0.5 g/L to about 5 g/L; or about 0.1 g/L to about 2 g/L. In some embodiments, the transposase polypeptide is present at about 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.2 g/L, 0.5 g/L, 1 , 2 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L, 35 g/L, 40 g/L, or 50 g/L.
In some embodiments, the suitable reaction conditions comprise a divalent metal cofactor. In some embodiments, the divalent metal cofactor is magnesium. In some embodiments, magnesium is present in the reaction mixture as magnesium (II) chloride. In some embodiments, the magnesium (II) chloride is present at concentrations of about 1 to 100 mM; about 1 to 50 mM; about 1 to 20 mM; or about 1 to 10 mM. In some embodiments, the magnesium (II) chloride is present at concentrations of about 3 mM; about 5 mM; about 7 mM, or about 10 mM.
During the course of the reaction, the pH of the reaction mixture may change. The pH of the reaction mixture may be maintained at a desired pH or within a desired pH range. This may be done by the addition of an acid or a base, before and/or during the course of the reaction. Alternatively, the pH may be controlled by using a buffer. Accordingly, in some embodiments, the reaction condition comprises a buffer. Suitable buffers to maintain desired pH ranges are known in the art and include, by way of example and not limitation, borate, phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N- morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine, and 2-amino-2-hydroxymethyl- propane-1 ,3-diol (Tris), and the like. In some embodiments, the reaction conditions comprise water as a suitable solvent with no buffer present.
In the embodiments of the process, the reaction conditions comprise a suitable pH. The desired pH or desired pH range can be maintained by use of an acid or base, an appropriate buffer, or a combination of buffering and acid or base addition. The pH of the reaction mixture can be controlled before and/or during the course of the reaction. In some embodiments, the suitable reaction conditions comprise a solution pH from about 4 to about 10, pH from about 5 to about 10, pH from about 5 to about 9, pH from about 6 to about 9, pH from about 6 to about 8. In some embodiments, the reaction conditions comprise a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.
In the embodiments of the processes herein, a suitable temperature is used for the reaction conditions, for example, taking into consideration the increase in reaction rate at higher temperatures, and the activity of the enzyme during the reaction time period. Accordingly, in some embodiments, the suitable reaction conditions comprise a temperature of about 10 °C to about 95 °C, about 10 °C to about 75 °C, about 15 °C to about 95 °C, about 20 °C to about 95 °C, about 20 °C to about 65 °C, about 25 °C to about 70 °C, or about 50 °C to about 70 °C. In some embodiments, the suitable reaction conditions comprise a temperature of about 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C or 95 °C. In some embodiments, the temperature during the enzymatic reaction can be maintained at a specific temperature throughout the course of the reaction. In some embodiments, the temperature during the enzymatic reaction can be adjusted over a temperature profile during the course of the reaction.
In some embodiments, the processes of the invention are carried out in a solvent. Suitable solvents include water, aqueous buffer solutions, organic solvents, polymeric solvents, and/or co-solvent systems, which generally comprise aqueous solvents, organic solvents and/or polymeric solvents. The aqueous solvent (water or aqueous co-solvent system) may be pH-buffered or unbuffered. In some embodiments, the processes using the engineered transposase polypeptides can be carried out in an aqueous co-solvent system comprising an organic solvent (e.g., ethanol, isopropanol (IPA), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) ethyl acetate, butyl acetate, 1 -octanol, heptane, octane, methyl t butyl ether (MTBE), toluene, and the like), ionic or polar solvents (e.g., 1 -ethyl 4 methylimidazolium tetrafluoroborate, 1 -butyl-3-methylimidazolium tetrafluoroborate, 1 -butyl 3 methylimidazolium hexafluorophosphate, glycerol, polyethylene glycols, and the like). In some embodiments, the co-solvent can be a polar solvent, such as a polyol, dimethylsulfoxide (DMSO), or lower alcohol. The non-aqueous co- solvent component of an aqueous co-solvent system may be miscible with the aqueous component, providing a single liquid phase, or may be partly miscible or immiscible with the aqueous component, providing two liquid phases. Exemplary aqueous co-solvent systems can comprise water and one or more co-solvents selected from an organic solvent, polar solvent, and polyol solvent. In general, the co-solvent component of an aqueous co-solvent system is chosen such that it does not adversely inactivate the transposase enzyme under the reaction conditions. Appropriate co-solvent systems can be readily identified by measuring the enzymatic activity of the specified engineered transposase enzyme with a defined substrate of interest in the candidate solvent system, utilizing an enzyme activity assay, such as those described herein.
In some embodiments of the process, the suitable reaction conditions comprise an aqueous cosolvent, where the co-solvent comprises DMSO at about 1% to about 50% (v/v), about 1 to about 40% (v/v), about 2% to about 40% (v/v), about 5% to about 30% (v/v), about 10% to about 30% (v/v), or about 10% to about 20% (v/v). In some embodiments of the process, the suitable reaction conditions can comprise an aqueous co-solvent comprising ethanol at about 1% (v/v), about 5% (v/v), about 10% (v/v), about 15% (v/v), about 20% (v/v), about 25% (v/v), about 30% (v/v), about 35% (v/v), about 40% (v/v), about 45% (v/v), or about 50% (v/v).
In some embodiments, the reaction conditions comprise a surfactant for stabilizing or enhancing the reaction. Surfactants can comprise non-ionic, cationic, anionic and/or amphiphilic surfactants. Exemplary surfactants, include by way of example and not limitation, nonyl phenoxypolyethoxylethanol (NP40), TRITON™ X-100 polyethylene glycol tert-octylphenyl ether, polyoxyethylene-stearylamine, cetyltrimethylammonium bromide, sodium oleylamidosulfate, polyoxyethylene-sorbitanmonostearate, hexadecyldimethylamine, etc. Any surfactant that may stabilize or enhance the reaction may be employed. The concentration of the surfactant to be employed in the reaction may be generally from 0.1 to 50 mg/mL, particularly from 1 to 20 mg/mL.
In some embodiments, the reaction conditions include an antifoam agent, which aids in reducing or preventing formation of foam in the reaction solution, such as when the reaction solutions are mixed or sparged. Anti-foam agents include non-polar oils (e.g., minerals, silicones, etc.), polar oils (e.g., fatty acids, alkyl amines, alkyl amides, alkyl sulfates, etc.), and hydrophobic (e.g., treated silica, polypropylene, etc.), some of which also function as surfactants. Exemplary anti-foam agents include Y-30® (Dow Corning), poly-glycol copolymers, oxy/ethoxylated alcohols, and polydimethylsiloxanes. In some embodiments, the anti-foam can be present at about 0.001% (v/v) to about 5% (v/v), about 0.01 % (v/v) to about 5% (v/v), about 0.1% (v/v) to about 5% (v/v), or about 0.1% (v/v) to about 2% (v/v). In some embodiments, the anti-foam agent can be present at about 0.001% (v/v), about 0.01% (v/v), about 0.1% (v/v), about 0.5% (v/v), about 1% (v/v), about 2% (v/v), about 3% (v/v), about 4% (v/v), or about 5% (v/v) or more as desirable to promote the reaction.
The quantities of reactants used in the transposase reaction will generally vary depending on the quantities of product desired, and concomitantly the amount of substrates employed. Those having ordinary skill in the art will readily understand how to vary these quantities to tailor them to the desired level of productivity and scale of production.
In some embodiments, the order of addition of reactants is not critical. The reactants may be added together at the same time to a solvent (e.g., monophasic solvent, biphasic aqueous co-solvent system, and the like), or alternatively, some of the reactants may be added separately, and some together at different time points. For example, the cofactor, co-substrate and substrate may be added first to the solvent.
The solid reactants (e.g., enzyme, salts, etc.) may be provided to the reaction in a variety of different forms, including powder (e.g., lyophilized, spray dried, and the like), solution, emulsion, suspension, and the like. The reactants can be readily lyophilized or spray dried using methods and equipment that are known to those having ordinary skill in the art. For example, the protein solution can be frozen at -80eC in small aliquots, then added to a pre-chilled lyophilization chamber, followed by the application of a vacuum.
For improved mixing efficiency when an aqueous co-solvent system is used, the transposase, and co-substrate may be added and mixed into the aqueous phase first. The substrate may be added and mixed in, followed by the organic phase or the substrate may be dissolved in the organic phase and mixed in. Alternatively, the substrate may be premixed in the organic phase, prior to addition to the aqueous phase.
The processes of the present invention are generally allowed to proceed until further conversion of substrate to product does not change significantly with reaction time (e.g., less than 10% of substrate being converted, or less than 5% of substrate being converted). In some embodiments, the reaction is allowed to proceed until there is complete or near complete conversion of substrate to product. Transformation of substrate to product can be monitored using known methods by detecting substrate and/or product, with or without derivatization. Suitable analytical methods include gas chromatography, HPLC, MS, and the like. In some embodiments, after suitable conversion to product, the reactants are separated from the product and additional reactants are added.
Any of the processes disclosed herein using the engineered polypeptides for the preparation of products can be carried out under a range of suitable reaction conditions, including but not limited to ranges of substrates, temperature, pH, solvent system, substrate loading, polypeptide loading, cofactor loading, and reaction time.
In some embodiments, the enzyme loading is between 1 -30% w/w. In some embodiments, additional reaction components or additional techniques are carried out to supplement the reaction conditions. These can include taking measures to stabilize or prevent inactivation of the enzyme, reduce product inhibition, shift reaction equilibrium to formation of the desired product.
In some embodiments, the engineered transposase polypeptides can be provided on a solid support, such as a membrane, resin, solid carrier, or other solid phase material. A solid support can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as co-polymers and grafts thereof. A solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica or metal, such as gold or platinum. The configuration of a solid support can be in the form of beads, spheres, particles, granules, a gel, a membrane or a surface. Surfaces can be planar, substantially planar, or non-planar. Solid supports can be porous or non-porous, and can have swelling or non-swelling characteristics. A solid support can be configured in the form of a well, depression, or other container, vessel, feature, or location.
In some embodiments, the engineered transposase polypeptides of the present invention can be immobilized on a solid support such that they retain their improved activity, and/or other improved properties relative to the reference polypeptide of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704. In such embodiments, the immobilized polypeptides can facilitate the biocatalytic conversion of the substrate compounds or other suitable substrates to the product and after the reaction is complete are easily retained (e.g., by retaining beads on which polypeptide is immobilized) and then reused or recycled in subsequent reactions. Such immobilized enzyme processes allow for further efficiency and cost reduction.
Methods of enzyme immobilization are well-known in the art. The engineered polypeptides can be bound non-covalently or covalently. Various methods for conjugation and immobilization of enzymes to solid supports (e.g., resins, membranes, beads, glass, etc.) are well known in the art (See e.g., Yi et al., Proc. Biochem., 42(5): 895-898 [2007]; Martin et al., Appl. Microbiol. Biotechnol., 76(4): 843-851 [2007]; Koszelewski et al., J. Mol. Cat. B: Enzymatic, 63: 39-44 [2010]; Truppo et al., Org. Proc. Res. Dev., published online: dx.doi.org/10.1021/op200157c; Hermanson, Bioconjugate Techniques, 2nd ed., Academic Press, Cambridge, MA [2008]; Mateo et al., Biotechnol. Prog., 18(3):629-34 [2002]; and “Bioconjugation Protocols: Strategies and Methods,” In Methods in Molecular Biology, Niemeyer (ed.), Humana Press, New York, NY [2004]; the disclosures of each which are incorporated by reference herein). Solid supports useful for immobilizing the engineered transposase of the present invention include but are not limited to beads or resins comprising polymethacrylate with epoxide functional groups, polymethacrylate with amino epoxide functional groups, styrene/DVB copolymer or polymethacrylate with octadecyl functional groups. Exemplary solid supports useful for immobilizing the engineered transposase polypeptides of the present invention include, but are not limited to, EnginZyme (including, EziG-1 , EziG- 1 , and EziG-3), chitosan beads, Eupergit C, and SEPABEADs (Mitsubishi) (including EC-EP, EC-HFA/S, EXA252, EXE1 19 and EXE120).
Methods are provided for using engineered transposases, i.e. , to reduce the complexity and time required for generating a mixture of altered nucleic acids or a plurality of nucleic acid fragments, i.e., for generating nucleic acid libraries from nucleic acid samples for nucleic acid sequencing, while simultaneously improving performance of the nucleic acid libraries. In some instances, the methods may be used for the generation of nucleic acid fragments (e.g., a sequence library) amenable to high throughput sequencing technologies. The engineered transposases are used, e.g., to insert excised transposable DNA elements for simultaneous fragmentation and tagging (e.g., barcoding) of a sample during sequence library preparation via the methods described herein.
In some instances, the methods described herein include contacting a target nucleic acid (e.g., nucleic acids of a nucleic acid sample) with a transposase (e.g., an engineered transposase) under conditions and for a time sufficient for the transposase to carry out a transposition event. In some instances, the transposition event occurs in reaction conditions additionally including a terminal deoxynucleotide transferase, dNTP, and/or buffer components suitable for the addition of deoxynucleotides to the 3’ terminus of each of the plurality of nucleic acid fragments. In some instances, the transposition event occurs in reaction conditions additionally including a DNA ligase and/or buffer components suitable for a ligation reaction.
In some instances, each altered nucleic acid of the mixture of altered nucleic acids is between 30 and 30000 (e.g., 30-30000, 30-25000, 30-20000, 30-15000, 30-10000, 30-7500, 30-5000, 30-4000, 30- 3000, 30-2500, 30-2000, 30-1500, 30-1000, 30-900, 30-800, 30-700, 30-600, 30-500, 30-400, 30-300, 30- 250, 30-200, 30-150, 30-100, 30-50, 100-500, 100-1000, 100-1500, 100-2500, 100-5000, 100-10000, 100-20000, 100-30000, 500-1000, 500-2500, 500-5000, 500-10000, 500-20000, 500-30000, 1000-2500, 1000-5000, 1000-10000, 1000-20000, 1000-30000, 5000-10000, 5000-20000, 5000-30000, 10000-20000, 20000-30000, 2500-5000, 7500-10000, or 15000-25000) nucleotides in length. In some instances, each altered nucleic acid of the mixture of altered nucleic acids further includes a sample tag and/or a unique molecular identifier (UMI). In some instances, each altered nucleic acid of the mixture of altered nucleic acids includes an identifiable sequence tag (1ST). In some instances, the 1ST is between 6 and 30 (e.g., 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length.
In some instances, each nucleic acid fragment of the plurality of nucleic acid fragments is between 30 and 30000 (e.g., 30-30000, 30-25000, 30-20000, 30-15000, 30-10000, 30-7500, 30-5000, 30- 4000, 30-3000, 30-2500, 30-2000, 30-1500, 30-1000, 30-900, 30-800, 30-700, 30-600, 30-500, 30-400, 30-300, 30-250, 30-200, 30-150, 30-100, 30-50, 100-500, 100-1000, 100-1500, 100-2500, 100-5000, 100- 10000, 100-20000, 100-30000, 500-1000, 500-2500, 500-5000, 500-10000, 500-20000, 500-30000, 1000- 2500, 1000-5000, 1000-10000, 1000-20000, 1000-30000, 5000-10000, 5000-20000, 5000-30000, 10000- 20000, 20000-30000, 2500-5000, 7500-10000, or 15000-25000) nucleotides in length. In some instances, each nucleic acid fragment of the subset of nucleic acid fragments further includes a sample tag and/or a unique molecular identifier (UMI). In some instances, each nucleic acid fragment of the subset of nucleic acid fragments includes an identifiable sequence tag (1ST). In some instances, the 1ST is between 6 and 30 (e.g., 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length.
Suitable samples and applications
Methods described herein (e.g., using engineered transposases) may be suitable for any samples and applications wherein sequencing technologies (e.g., next-generation sequencing (NGS)) may be performed. A sample may derive from a single cell or a plurality of single cells. A sample may derive from a whole population of cells or from a larger cell population. A sample may derive from one cell type or a plurality of cell types. A sample may derive from bacteria, fungi, viruses, animal tissue, or plant tissue. A sample may be isolated or partially isolated. One or more samples may be used for applications including but not limited to whole genome sequencing, whole exome sequencing, transcriptome analysis, epigenome sequencing, sequencing of target regions, deep sequencing, among other clinical and research applications.
A sample may be one or more nucleic acids of interest that may be used to generate a sequence library suitable for NGS or similar sequencing methods. A nucleic acid may be DNA (e.g., genomic, mitochondrial DNA, or a combination thereof), RNA (e.g., mRNA, rRNA, tRNA, microRNA, non-coding RNA, or a combination thereof), or a combination of both DNA and RNA. In some instances, an RNA sample is converted to complementary DNA (cDNA) using enrichment and reverse transcription methodologies known in the art. In some instances, an RNA sample can be transformed into an RNA/DNA duplex by reverse-transcription using a suitable reverse transcriptase, including, e.g., a Moloney murine leukemia virus (M-MLV) reverse transcriptase, a human immunodeficiency virus (HIV) reverse transcriptase, and an avian sarcoma-leukosis virus (ASLV) reverse transcriptase. Avian Sarcoma- Leukosis Virus (ASLV) reverse transcriptase includes, but is not limited to, Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV reverse transcriptase, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A reverse transcriptase, Avian Sarcoma Virus UR2 Helper Virus UR2AV reverse transcriptase, Avian Sarcoma Virus Y73 Helper Virus YAV reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, and Myeloblastosis Associated Virus (MAV) reverse transcriptase. In some instances, a nucleic acid may include genomic DNA or cDNAs from a single cell. In some instances, a nucleic acid may include nucleic acids from a plurality of haplotypes. In some instances, a nucleic acid may include crosslinked via histones or chromatin from single or multiple cells. In some instances, a nucleic acid may include nucleic acid that has been condensed or optionally treated with one or more condensing agents.
Sequence Library Construction
Provided are methods using transposases with improved activity in cleaving or fragmenting polynucleotides and ligating or tagging polynucleotides (e.g., the engineered transposases described herein) that overcome the limitations of existing NGS library preparation methods. Improved transposases with increased polynucleotide fragmentation or cleavage activity, increased thermostability, increased activity at elevated temperatures, increased soluble expression, decreased inhibition, increased adapter loading or adapter ligation (i.e. tagging), increased binding to a target polynucleotide, and/or decreased sequence insertion bias compared to another wild-type or engineered transposase find utility in library preparation for NGS, but are not limited to these uses. Improved transposases (e.g., the engineered transposases described herein) can find use in any application involving cleaving or fragmenting doublestranded polynucleotides and/or ligating adapters or tagging polynucleotides, including applications where a donor polynucleotide is transferred into a target polynucleotide.
The present methods employ novel transposases that have improved activity in the fragmentation or cleavage of double stranded DNA and the ligation of adapters or tagging of the DNA fragments. The transposases employed in the methods have increased polynucleotide fragmentation or cleavage activity, increased thermostability, increased activity at elevated temperatures, increased soluble expression, decreased inhibition, increased adapter loading or adapter ligation (i.e. tagging), increased binding to a target polynucleotide, and/or decreased sequence insertion bias as compared to another wild-type transposase or engineered transposase. The engineered polypeptides employed in the methods are variants of SEQ ID NO: 2 or SEQ ID NO: 5704, a chimeric protein based an IS4 transposase from Parashewanella curva or a modified Escherichia coli Tn5 transposase, respectively, which recognize the19 bp Tn5 mosaic end (ME) transposon donor recognition sequence. These engineered transposases are capable of improved preparation of NGS libraries from polynucleotide samples.
In nature, transposases facilitate the transposition of fragments of DNA (transposons) from a donor strand to a host strand at sites flanked by short inverted-repeat sequences or end sequences (ESs). The transposition mechanism is initiated by the binding of two transposases to donor DNA ESs to form the dimeric synaptic complex, which catalyzes cleavage of the donor DNA. The synaptic complex (i.e., transposome; dimeric transposase/DNA complex) then binds to the target DNA to transfer the transposon DNA to the target DNA.
Transposase-mediated NGS library preparation is similar to naturally-occurring transposition, except that the donor DNA or polynucleotide consists of two, separate adapter sequences rather than one continuous loop of donor DNA or polynucleotide. These adapter sequences (e.g., i5 and i7 adapter oligonucleotides) are bound to transposases in a loading step that replaces the initial donor DNA or polynucleotide binding and cleavage step. Following adapter loading, the dimerized synaptic complex (i.e. , transposome) proceeds to bind the target DNA or polynucleotide sample. Cleavage of the target polynucleotide and ligation of the adapter sequences results in fragmentation of the target DNA or polynucleotide and ligation of sequencing adapters to the target fragments.
The resulting DNA or polynucleotide fragments with ligated adapters form the next generation sequencing library. An ideal library preparation method would generate random fragments in the polynucleotide sequence, without bias toward the sequence composition of the target site. Compared to traditional library preparation methods, transposase-mediated fragmentation has lower sequence insertion bias. However, certain transposases are known to have sequence insertion bias. As an example, Tn5 has a slight G/C sequence insertion bias. In some instances, the engineered transposases employed in the methods have reduced sequence insertion bias or increased sequence promiscuity as compared to a wild-type or reference transposase.
The methods described herein can be used for the generation of nucleic acid libraries having amplicons using adapter oligonucleotides and barcoded amplifier oligonucleotides. In some instances, the methods include generating the nucleic acid libraries using (a) a DNA polymerase; (b) a first synaptic complex including a first transposase (e.g., an engineered transposase described herein) and a first adapter oligonucleotide, and a second synaptic complex including a second transposase (e.g., an engineered transposase described herein) and a second adapter oligonucleotide; and (c) magnesium ions (e.g., Mg2+). In some instances, each adapter oligonucleotide includes an adapter priming region. The method may further include using (i) a first amplifier oligonucleotide including a first universal primer region, a first amplifier barcode region (e.g., a pool tag, e.g., an i5 or an 17 tag), and a first amplifier priming region; and (ii) a second amplifier oligonucleotide including a second universal primer region, a second amplifier barcode region (e.g., a pool tag, e.g., an i5 or an 17 tag), and a second amplifier priming region, wherein the first adapter priming region of the first adapter oligonucleotide is homologous to the first amplifier priming region of the first amplifier oligonucleotide and the second adapter priming region of the second adapter oligonucleotide is homologous to the second amplifier priming region of the second amplifier oligonucleotide. In some instances, each adapter oligonucleotide includes a universal primer region and an adapter barcode region (e.g., an identifiable sequence tag (1ST) or a sample tag).
T ansposition
The nucleic acid samples of a sequencing library (e.g., a NGS library) may be generated in a single reaction vessel through a method that includes a transposition reaction (e.g., using engineered transposases described herein), a polymerization reaction, and a PCR reaction for a tagmentation-based sequence library preparation. The method includes first combining the DNA polymerase, the first synaptic complex and the second synaptic complex, magnesium ions (e.g., Mg2+), the first and second amplifier oligonucleotides, and the nucleic acid sample in a first reaction vessel. The engineered transposases of the synaptic complexes as described herein will fragment the nucleic acid sample into nucleic acid fragments and add the adapter oligonucleotide sequences to the 5’ ends of the two strands of a nucleic acid duplex containing a nucleic acid fragment and its complement sequence in a transposition reaction to generate intermediate nucleic acids. In some instances, the transposition reaction occurs at a transposition reaction temperature between 25-65 °C (e.g., between 35-65 °C, between 40-65 °C, between 45-65 °C, between 50-65 °C, between 55-65 °C, between 60-65 °C, between 25-60 °C, between 25-55 °C, between 25-50 °C, between 25-45 °C, between 25-40 °C, between 25-35 °C, between 25-30 °C, between 40-50 °C, or between 53-57 °C; e.g., at about 25 °C, at about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 53 °C, about 54 °C, about 55 °C, about 56 °C, about 57 °C, about 60 °C, or about 65 °C). In some instances, a transposition reaction temperature is selected following consideration of the rate of reaction and transposase activity at a given temperature or range of temperatures. In some instances, a specific temperature is maintained over the course of the enzymatic reaction to maximize enzymatic activity or reaction efficiency. In other instances, the temperature increases or decreases over a range of temperatures to maximize enzymatic activity or reaction efficiency.
In some instances, the transposition reaction occurs for a first reaction duration between 1 and 30 minutes (e.g., between 1 and 25 minutes, between 1 and 20 minutes, between 1 and 15 minutes, between 1 and 10 minutes, between 1 and 5 minutes, between 5 and 10 minutes, between 5 and 20 minutes, between 5 and 30 minutes, between 10 and 30 minutes, between 15 and 30 minutes, between 20 and 30 minutes, between 25 and 30 minutes, between 10 and 20 minutes, between 15 and 25 minutes; e.g., about 1 minute, about 5 minutes, about 10 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 20 minutes, about 25 minutes, or about 30 minutes). In some instances, the duration of the transposition reaction is determined by the temperature or range of temperatures at which the transposition reaction occurs. In some instances, the duration of the transposition reaction is determined by the expected efficiency or stability of a transposase.
In some instances, the nucleic acid sample for fragmentation is present in the transposition reaction at variable amounts. The sample may be present at low concentrations at about 1 to 10 ng/pL (e.g., about 10 ng/pL, about 9 ng/pL, about 8 ng/pL, about 7 ng/pL, about 6 ng/pL, about 5 ng/pL, about 4 ng/pL, about 3 ng/pL, about 2 ng/pL, about 1 ng/pL, or less than 1 ng/pL). The sample may be present at medium concentrations at about 10 ng/pL to 100 ng/pL (e.g., about 100 ng/pL, about 90 ng/pL, about 80 ng/pL, about 70 ng/pL, about 60 ng/pL, about 50 ng/pL, about 40 ng/pL, about 30 ng/pL, about 20 ng/pL, or about 10 ng/pL). Further, the sample may be present at high concentrations at about 100 ng/pL or higher (e.g., about 100 ng/pL, about 150 ng/pL, about 200 ng/pL, about 250 ng/pL, about 300 ng/pL, or higher). For each of the sample tagging reaction concentration ranges, a relatively fixed and limited sample-tagging condition is used such that the number of tags (e.g., sample tags or identifiable sequence tags (ISTs)) per prescribed unit mass of nucleic acid is limited. The resulting tagmented samples thereby have a similar number of tagmentation occurrences but still a varying amount of nucleic acid molecules. The plurality of tagged DNA samples is pooled to create a sample pool in which a multiplicity of potentially differently tagged DNA samples produced by transposition processes are combined into one heterogeneous mixture. The important feature of this pool is that while it may include different masses of nucleic acid molecules from each sample in rough proportion to the mass of nucleic acid molecules in each sample, the number of sample tagging instances from each sample present in the pool is fixed and normalized.
In some instances, one or more incorporated tags may consist of the same known oligonucleotide sequence, or a different oligonucleotide sequence. One or more tags may consist of oligonucleotide sequences of the same lengths or different lengths, wherein said lengths range from 4 to 10 base pairs (e.g., 4 base pairs, 5 base pairs, 6 base pairs, 7 base pairs, 8 base pairs, 9 base pairs, or 10 base pairs), 8 to 15 base pairs (e.g., 8 base pairs, 9 base pairs, 10 base pairs, 11 base pairs 12 base pairs, 13 base pairs, 14 base pairs, or 15 base pairs), or 10 to 20 base pairs (e.g., 10 base pairs, 11 base pairs, 12 base pairs, 13 base pairs, 14 base pairs, 15 base pairs, 16 base pairs, 17 base pairs, 18 base pairs, 19 base pairs, or 20 base pairs).
Polymerization
After the transposition reaction, a polymerization reaction by a polymerase (e.g., a DNA polymerase) can extend the 3’ ends of the intermediate nucleic acids to generate full duplexes. In some instances, the polymerization reaction occurs at a polymerization reaction temperature between 55-95 °C (e.g., between 60-95 °C, between 65-95 °C, between 70-95 °C, between 75-95 °C, between 80-95 °C, between 85-95 °C, between 90-95 °C, between 55-90 °C, between 55-85 °C, between 55-80 °C, between 55-75 °C, between 55-70 °C, between 55-65 °C, between 55-60 °C, between 65-85 °C, between 70-80 °C, between 73-77 °C; e.g., at about 55 °C, at about 60 °C, at about 65 °C, at about 70 °C, at about 73 °C, at about 74 °C, at about 75 °C, at about 76 °C, at about 77 °C, at about 80 °C, at about 85 °C, at about 90 °C, at about 95 °C). In some instances, the temperature of the polymerization reaction is dependent on the enzymatic activity and efficiency of the DNA polymerase. In some instances, the transposases dissociate as the first reaction vessel is heated to the polymerization temperature. In some instances, the DNA polymerase may be a thermostable DNA polymerase. In some instances, the DNA polymerase may be a high-fidelity DNA polymerase. In some instances, the DNA polymerase may be a hot-start DNA polymerase. In some instances, the hot-start DNA polymerase may contain an antibody or aptamer bound to the DNA polymerase, that is released when the hot-start DNA polymerase heated to and incubated at a suitable temperature, which may or may not be higher than the optimal polymerization temperature. In some instances, the DNA polymerase is selected based on the GC content of the sample or desired coverage following amplification.
In some instances, the polymerization reaction occurs for a second reaction duration between 1 and 60 minutes (e.g., between 1 and 55 minutes, between 1 and 50 minutes, between 1 and 45 minutes, between 1 and 40 minutes, between 1 and 35 minutes, between 1 and 30 minutes, between 1 and 25 minutes, between 1 and 20 minutes, between 1 and 15 minutes, between 1 and 10 minutes, between 10 and 60 minutes, between 15 and 60 minutes, between 20 and 60 minutes, between 25 and 60 minutes, between 30 and 60 minutes, between 35 and 60 minutes, between 40 and 60 minutes, between 45 and 60 minutes, between 50 and 60 minutes, between 55 and 60 minutes, between 15 and 35 minutes, between 30 and 50 minutes, between 10 and 20 minutes, between 20 and 40 minutes, or between 13 and 17 minutes; e.g., about 1 minute, about 5 minutes, about 10 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes). In some instances, the duration of the polymerization reaction is dependent on the temperature or range of temperatures at which the polymerization reaction occurs. Amplification
In some instances, nucleic acid samples (e.g., nucleic acid fragment) may be amplified through polymerase chain reaction (PCR), multiple displacement amplification (MDA), ligase chain reaction (LCR), loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), or strand displacement amplification (SDA).
For example, a PCR reaction generates the amplicons of the sequence library by using the amplifier oligonucleotides as primers. In some instances, the amplicons are generated in a PCR reaction with a first universal primer and a second universal primer, wherein the first universal primer and the second universal primer bind to respective complementary universal primer regions in the sequencing oligonucleotides. In some instances, the amplicons may be further amplified in a PCR reaction with a first universal primer and a second universal primer, wherein the first universal primer and the second universal primer bind to respective complementary universal primer regions in the amplicons.
In some instances, the amplicons include (a) a nucleic acid sequence including a first universal primer region, a first amplifier barcode region (e.g., a pool tag, e.g., an i5 or an i7 tag), a first amplifier priming region, a homologous sequence of a first nucleic acid fragment, a complement sequence of the second amplifier priming region, a complement sequence of the second amplifier barcode region (e.g., a pool tag, e.g., an i5 or an i7 tag), and a complement sequence of the second universal primer region; and (b) the complement sequence thereof. In some instances, a nucleic acid duplex including the first nucleic acid fragment and its complement is generated from the nucleic acid sample by transposase activity. In some instances, the intermediate nucleic acid is the template for the PCR reaction. In some instances, the PCR reaction produces amplicons from the intermediate nucleic acids using the amplifier oligonucleotides. In some instances, the PCR reaction includes 1 -35 cycles (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 cycles). In some instances, the PCR reaction includes more than 35 cycles. In some instances, the PCR reaction may be monitored by tracking the cycle threshold (Ct) via fluorescence detection using well- established methods in the art. In some instances, the resulting sequence library following transposition, tagmentation, and amplification may be evaluated for quality (e.g., size distribution, purity, concentration, among other quality considerations). In some instances, the sequence library may be assessed for the size distribution of the resulting fragments by gel electrophoresis compared to a reference sample (e.g., a DNA ladder).
Reaction Considerations
In the instances provided herein, various ranges of suitable reaction conditions that can be used in the processes, include but are not limited to, substrate loading, co-substrate loading, pH, temperature, buffer, solvent system, cofactor, polypeptide loading, and reaction time. Further suitable reaction conditions for carrying out the process for biocatalytic conversion of nucleic acid samples to a sequencing library and its intermediates using an engineered transposase described herein can be readily optimized in view of the guidance provided herein by routine experimentation that includes, but is not limited to, contacting the transposase and one or more substrate compounds (e.g., nucleic acid samples; e.g., DNA) under experimental reaction conditions of concentration, pH, temperature, and solvent conditions, and detecting the product compound.
In some instances, one or more enzymes (e.g., a reverse transcriptase, a transposase (e.g., an engineered transposase described herein), or a DNA polymerase) are inactivated by the application of heat or an inhibitor following a chemical reaction (e.g., transposition, polymerization, or amplification) in the tagmentation-based sequence library preparation. In some instances, one or more enzymes are inactivated by changes in buffer or solvent condition (e.g., changes in pH or changes in salt concentration). In some instances, the nucleic acid sample is purified or partially purified following a chemical reaction. In some instances, the nucleic acid sample is concentrated or diluted following a chemical reaction.
In some instances, one or more enzymes (e.g., a reverse transcriptase, a transposase (e.g., an engineered transposase described herein), or a DNA polymerase) are immobilized to improve reaction efficiency, enzyme activity, or sample recovery. In some instances, an enzyme is immobilized on a solid support, a surface, a resin, a membrane, a bead, a nanoparticle, a biological cofactor (e.g., a protein, an antibody or antibody fragment, a peptide, an oligonucleotide, an aptamer), or a combination thereof. In some instances, an enzyme is immobilized through an irreversible or a reversible reaction. In some instances, an enzyme is immobilized by cross-linking, adsorption, entrapment, encapsulation, click chemistry reactions, ionic bonding, covalent bonding, hydrophobic interactions, among other non-limiting methods of immobilization.
In some instances, the nucleic acid sample, the first adapter oligonucleotide, the second adapter oligonucleotide, the first amplifier oligonucleotide, the second amplifier oligonucleotide, the intermediate nucleic acids, and/or the amplicons may include DNA. In some instances, the nucleic acid sample may include double-stranded DNA.
In some instances, further processing steps are used for library preparation, including gap filling, normalization, pooling of tagged fragment samples, and/or ligation of additional adapters to create dualtagged libraries. A variety of parameters may be manipulated to create sequencing libraries with various characteristics. For example, varying the concentration of transposase and adapters can change the fragment length. Copy number and sample coverage may also be influenced or controlled using methods known to those of skill in the art.
Methods of Sequencing
Methods described herein can be used for determining the nucleic acid sequences of the sequencing oligonucleotides or amplicons through nucleic acid sequencing (e.g., next-generation sequencing (NGS)) or other methods known in the art. In some instances, sequencing can be performed by various systems that are currently available, e.g., a sequencing system by Pacific Biosciences (PACBIO®), ILLUMINA®, Oxford NANOPORE®, Genapsys®, or ThermoFisher (ION TORRENT®). Alternatively or in addition, sequencing may be performed using nucleic acid amplification, sequencing by ligation (e.g., SOLiD or polony-based sequencing), sequencing by synthesis (e.g., Illumina dye sequencing, single-molecule real-time (SMRT) sequencing, or pyrosequencing), nanopore sequencing, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR), or isothermal amplification. In some instances, the sequencing oligonucleotides and amplicons described herein can be uniquely identified based on the nucleic acid sequences of the nucleic acid fragment and the nucleic acid sequences of the adapter barcode regions (e.g., an identifiable sequence tag (1ST) or a sample tag) or amplifier barcode regions (e.g., pool tags, e.g., i5 or 17 tags) of the sequencing oligonucleotides or amplicons, respectively. The invention further includes data generated by nucleic acid sequencing, as well as methods for generating and analyzing such sequence data, and reaction mixtures used in and formed by such methods.
Methods of Evaluating Transposition Activity
Methods described herein can be used for assaying transposition activity of a transposase described herein (e.g., an engineered transposase described herein. In some instances, the methods include performing a transposition reaction on a nucleic acid (e.g., DNA, e.g., double-stranded DNA (dsDNA)), i.e. , to insert a nucleic acid sequence of interest (e.g., DNA, e.g., dsDNA, e.g., dsDNA adapters) into a target nucleic acid sequence (e.g., DNA, e.g., dsDNA). In some instances, synaptic complexes (i.e., transposomes) are prepared by incubating transposases (e.g., engineered transposases described herein) with nucleic acid adapters (e.g., DNA adapter oligonucleotides). In some instances, 1 - 50 pM (e.g., 1 -40, 1 -30, 1 -20, 1 -10, 1 -5, 5-50, 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20, 20-50, 20-40, or 20-30 pM; e.g., about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 pM) of the adapter may be incubated with engineered transposases for about 60 minutes to load the transposases and generate synaptic complexes. In some instances, a target substrate (e.g., a target dsDNA) is added and incubated with the synaptic complexes until a transposition event occurs. In some instances, the mixture of the synaptic complexes and the target substrate is incubated (e.g., heated in a thermocycler). In some instances, the mixture is incubated at about 55 °C for 5-15 minutes (e.g., about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 minutes). In some instances, the target substrate is any of the nucleic acid samples described herein, including DNA or cDNA, and may be prokaryotic, eukaryotic, or artificial in origin.
In some instances, the activity of two different transposases (e.g., any transposases described herein or known to one of skill in the art, e.g., any naturally occurring transposases or any engineered transposases described herein), i.e., a first transposase and a second transposase, may be compared by comparing the amount of transposition reactions that occur when using synaptic complexes comprising the first transposase versus synaptic complexes comprising the second transposase. Preferably, the other reaction conditions are maintained similar or identical for the comparison (e.g., target nucleic acid concentration, reaction temperature, reaction duration, adapter concentration, transposase concentration, buffer conditions). In some instances, an activity fold improvements over positive control (FIOP) can be calculated as the percent product of transposition reactions produced by the first transposase (e.g., an engineered transposase) compared with the percent product of transposition reactions produced by the second transposase (e.g., a reference transposase), i.e., as an “FIOP Percent Product.” Methods of Evaluating Transposition Performance
Methods described herein can be used for assaying transposition performance of a transposase employed in the methods (e.g., an engineered transposase described herein. In some instances, the methods include generating an NGS library from a nucleic acid sample (e.g., DNA, e.g., double-stranded DNA (dsDNA)), i.e., by performing a tagmentation reaction (e.g., to fragment the nucleic acid sample and insert adapter nucleic acids DNA, e.g., dsDNA, e.g., dsDNA adapters) and amplifying the nucleic acid fragments resulting from the tagmentation reaction. In some instances, synaptic complexes (i.e., transposomes) are prepared by incubating transposases (e.g., engineered transposases described herein) with nucleic acid adapters (e.g., DNA adapter oligonucleotides, e.g., i5 and i7 oligonucleotides) to generate loaded i5 and 17 transposases. In some instances, a nucleic acid sample is mixed with the synaptic complexes (e.g., loaded i5 and 17 transposases) and incubated until a transposition reaction occurs. In some instances, the mixture is incubated at about 55 °C for 5-15 minutes (e.g., about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 minutes). In some instances, the transposition reaction is performed using a method described herein. In some instances, the product of the transposition reaction is amplified, e.g., using a method of nucleic acid amplification known in the art (e.g., a PCR reaction, e.g., using primers suitable for the i5 and I7 adapter sequences). In some instances, the nucleic acid sample is any of the nucleic acid samples described herein, including DNA or cDNA, and may be prokaryotic, eukaryotic, or artificial in origin. In some instances, the NGS library is performed using methods described herein, e.g., in the section above titled “Sequence Library Construction.”
In some instances, the performance of two different transposases (e.g., any transposases described herein or known to one of skill in the art, e.g., any naturally occurring transposases or any engineered transposases described herein), i.e., a first transposase and a second transposase, may be compared by comparing the insertion bias (e.g., sequence insertion bias, e.g., G/C bias) of transposition reactions that occur when using synaptic complexes comprising the first transposase versus synaptic complexes comprising the second transposase. Preferably, the other reaction conditions are maintained similar or identical for the comparison (e.g., target nucleic acid concentration, reaction temperature, reaction duration, adapter concentration, transposase concentration, buffer conditions). In some instances, an activity fold improvements over positive control (FIOP) can be calculated as the percent product of transposition reactions produced by the first transposase (e.g., an engineered transposase) compared with the percent product of transposition reactions produced by the second transposase (e.g., a reference transposase), i.e., as an “FIOP Percent Product.” In some instances, a FIOP can be calculated as the relative calculated enzyme activity of the first transposase (e.g., an engineered transposase) compared to the second transposase (e.g., a reference transposase), such as by comparing fragmenting activity of a nucleic acid sequence (e.g., a double-stranded nucleic acid sequence) and/or transposition of a sequence.
In some instances, transposase promiscuity or target bias of a first transposase (e.g., an engineered transposase) relative to a second transposase (e.g., a reference transposase) can be calculated as the ratio of the product peak area produced by the first transposase (e.g., the product of a first pair of annealed oligos vs. product of a second pair of annealed oligos (e.g., pairs of annealed oligonucleotides comprising a polynucleotide sequence selected from SEQ ID NOs: 2370-2387 and 5696- 5700)) divided by the ratio of the product peak area observed by the reaction with the second transposase (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate), e.g., as a “Promiscuity FIOP” or “FIOP Target Product Ratio.”
In some instances, transposon insertion frequency is biased by the sequence of the insertion sites over the first 15 nucleotides of NGS reads putatively comprising the region of the target bound by the transposome construct. In some instances, bias plots can be generated from NGS data to represent this bias as a function of the distance from the insertion site. In some instances, overall relative bias for each transposase (e.g., an engineered transposase) can be calculated as the root-mean-squared deviation (RMSD) of the observed vs. expected representation of all four nucleotides at each position as shown in Scheme 1 . Additionally, the mean RMSD over, for example, the first 15 nucleotides (RMSD15) can be used to summarize sequence insertion bias with a single number.
Scheme 1.
In some instances, relative target bias for each transposase (e.g., engineered transposase) can be calculated as the mean RMSD15 of a second transposase (e.g., a reference transposase) divided by the mean RMSD15 of a first transposase (e.g., an engineered transposase). In this manner, engineered transposases with target bias mean RMSD15 less than a reference transposase will have a ratio greater than 1 .
In further embodiments, any of the above described processes for the conversion of one or more substrate compounds to product compound can further comprise one or more steps selected from: extraction; isolation; purification; and crystallization of product compound. As is known to those skilled in the art, acidic compounds may exist in various salt forms that can be used interchangeably in the methods described herein. All such forms are specifically envisaged for use in the methods described herein. Methods, techniques, and protocols for extracting, isolating, purifying, and/or crystallizing the product from biocatalytic reaction mixtures produced by the above disclosed processes are known to the ordinary artisan and/or accessed through routine experimentation. Additionally, illustrative methods are provided in the Examples below.
Various features and embodiments of the invention are illustrated in the following representative examples, which are intended to be illustrative, and not limiting.
EXAMPLES
The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention. Indeed, there are various suitable sources for many of the reagents and equipment described below. It is not intended that the present invention be limited to any particular source for any reagent or equipment item.
In the experimental disclosure below, the following abbreviations apply: M (molar); mM (millimolar), uM and pM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and pg (micrograms); L and 1 (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters); urn and pin (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations per minute); psi and PSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); CV (coefficient of variability); CAM and cam (chloramphenicol); PMBS (polymyxin B sulfate); IPTG (isopropyl p-D-l-thiogalactopyranoside); LB (lysogeny broth); TB (terrific broth); SFP (shake flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); nt (nucleotide; polynucleotide); aa (amino acid; polypeptide); E. coli W31 10 (commonly used laboratory E. coli strain, available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high pressure liquid chromatography); HPLC-UV (HPLC-Ultraviolet Visible Detector); 1 H NMR (proton nuclear magnetic resonance spectroscopy); FIOPC (fold improvements over positive control); Sigma and Sigma- Aldrich (Sigma-Aldrich, St. Louis, MO; Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, Ml); Microfluidics (Microfluidics, Westwood, MA); Life Technologies (Life Technologies, a part of Fisher Scientific, Waltham, MA); Amresco (Amresco, LLC, Solon, OH); Carbosynth (Carbosynth, Ltd., Berkshire, UK); Varian (Varian Medical Systems, Palo Alto, CA); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Infors (Infors USA Inc., Annapolis Junction, MD); and Thermotron (Thermotron, Inc., Holland, Ml).
EXAMPLE 1
Transposase Gene Acquisition and Construction of Expression Vectors
The wild-type (WT) polypeptide described by UniProt accession number A0A3L8PTF2 from species Parashewanella curva is a predicted IS4 family transposase. A synthetic gene (SEQ ID NO: 1 ) encoding a C-terminal 6-histidine tagged version of the transposase was designed in silico to recognize the 19 bp Tn5 mosaic end (ME) transposon donor recognition sequence with codon optimization for E. coli expression, was synthesized, and was subcloned into the E. co// expression vector pCK100900i (See e.g., US Pat. No. 7,629,157 and US Pat. Appln. Publn. 2016/0244787, both of which are hereby incorporated by reference). This plasmid construct was transformed into an E. coli strain derived from BL-21 . Directed evolution techniques were used to generate libraries of gene variants from these plasmids (See e.g., US Pat. No. 8,383,346 and WO 2010/144103, both of which are hereby incorporated by reference).
EXAMPLE 2
Transposase Expression and Lysate Processing for High Throughput (HTP) Screening High Throughput (HTP) Growth of Transposase Enzyme and Variants
Transformed E. co// cells were selected by plating onto LB agar plates containing 1 % glucose and 30 pg/mL chloramphenicol. After overnight incubation at 37°C, colonies were placed into the wells of 96- well shallow flat bottom NUNC™ (Thermo-Scientific) plates filled with 190 pl/well LB medium supplemented with 1 % glucose and 30 pg/mL chloramphenicol. The cultures were allowed to grow overnight for 18-20 hours in a shaker (200 rpm, 30°C, and 85% relative humidity; Kuhner). Overnight growth samples (20 pL) were transferred into Costar 96-well deep plates filled with 380 pL of Terrific Broth supplemented with 30 pg/mL chloramphenicol. The plates were incubated for 120 minutes in a shaker (250 rpm, 30°C, and 85% relative humidity; Kuhner) until the ODeoo reached between 0.4-0.8. The cells were then induced with 40 pL of 10 mM IPTG in sterile water and incubated overnight for 18-20 hours in a shaker (250 rpm, 20-30°C, and 85% relative humidity; Kuhner). The cells were pelleted (4,000 rpm for 20 min), the supernatants were discarded, and the cells were frozen at -80 °C prior to analysis.
For lysis, 300 pL lysis buffer containing 1 g/L lysozyme were added to the cell pellet in each well. The cells were resuspended by vigorous shaking at room temperature for 30-90 minutes on a bench top shaker. A 150-uL aliquot of the re-suspended cells was transferred to a 96-well format 200 pL BioRad PCR plate and heated at the lysis temperature from 30-60°C for 30 mins as described in the examples below. Following heat-treatment, the cell debris was pelleted by centrifugation (4,000 rpm, 4°C, 10 mins) and clear supernatants were then used in biocatalytic reactions to determine their activity levels.
EXAMPLE 3
Shake Flask Expression and Purification of Transposase
Shake Flask Expression
Selected HTP cultures grown as described above were plated onto LB agar plates with 1 % glucose and 30 pg/mL chloramphenicol and grown overnight at 37eC. A single colony from each culture was transferred to 5 mL of LB broth with 1 % glucose and 30 pg/mL chloramphenicol. The cultures were grown for 20 h at 30 °C, 250 rpm, and subcultured at a dilution of approximately 1 :50 into 250 mL of Terrific Broth with 30 pg/mL of chloramphenicol. The cultures were incubated for approximately 3-4 hours at 30 °C, 250 rpm, to an ODeoo of about 0.6-0.8, and then induced with the addition of IPTG at a final concentration of 1 mM. The induced cultures were incubated for 18-20 h at 20°C-30°C, 250 rpm. Following this incubation period, the cultures were centrifuged at 4,000 rpm for 10 min. The culture supernatant was discarded, and the pellets were resuspended in 20 mL of 50 mM Tris, 500 mM NaCI, 20 mM Imidazole, pH 8.0. This cell suspension was chilled in an ice bath and lysed using a Microfluidizer cell disruptor (Microfluidics M-1 10L). The crude lysate was pelleted by centrifugation (10,000 rpm for 60 mins at 4 °C), and the supernatant was then filtered through a 0.2 pm PES membrane to further clarify the lysate.
Purification of Transposase from Shake Flask Lysates
Transposase lysates were purified using an AKTA Start purification system and a 5mL HisTrap FF column (GE Healthcare) using the AC Step HiF setting (the run parameters are provided below). The shake flask purification wash buffer comprised of 50 mM Tris-HCI, 500 mM NaCI, 20 mM imidazole, pH 8.0. The shake flask purification elution buffer comprised of 50 mM Tris-HCI, 500 mM NaCI, 250 mM imidazole, pH 8.0.
Elution fractions containing protein were identified by UV absorption (A280) and pooled, then dialyzed overnight in dialysis buffer (50 mM Tris, 150 mM NaCI, 0.1 mM EDTA, 50% glycerol, pH 8.0) in a 3.5K Slide-A-Lyzer™ dialysis cassette (Thermo Fisher) for buffer exchange. Transposase concentrations in the preparations were measured by absorption at 280 nm.
EXAMPLE 4
Capillary Electrophoresis (CE) Analysis of Oligonucleotides
Sample preparation for reaction analysis using CE:
For analysis of the assay reaction samples, capillary electrophoresis was performed using an ABI 3500xL or SeqStudio 24 Flex Genetic Analyzers (ThermoFisher). 2 pL of the final diluted assay samples were further diluted 10x in a new 96-well MicroAmp Optical PCR plate or 384-well MicroAmp Optical PCR plate with 18 pL of Hi-Di™ Formamide (ThermoFisher) containing an appropriate size standard (LIZ or Alexa633).
CE analysis was done using POP6 polymer and a capillary length of 50 cm. Oven temperature was set at 50°C. The prerun, run and injection voltages were 18, 19.5 and 5 kVolts respectively. The prerun, run and injection times were 180, 850 and 5 secs. FAM-labeled oligo substrates (forward/reverse [odd/even] annealed oligos summarized in Table 4.1) and products were identified by their sizes relative to the sizing ladder.
EXAMPLE 5 Improvements Over SEQ ID NO: 2 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 2 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2. Activity of the transposase variants was determined using a capillary electrophoresis (CE) assay as shown in Figure 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter supplemented with 7 mM MgCl2 at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 5.1 . Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 5.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 2 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 2 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 5.2.
EXAMPLE 6
Improvements Over SEQ ID NO: 4 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 4 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 6.1 . Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 6.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 4 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 4 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 6.2.
EXAMPLE 7
Improvements Over SEQ ID NO: 156 in the Transposition of Double-Stranded DNA (dsDNA) Adapters Into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 156 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2. Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 7.1 . Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 7.1 , and analyzed on CE as described in Example 4. Activity relative to SEQ ID NO: 156 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380 produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380 observed by the reaction with SEQ ID NO: 156 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 7.2. EXAMPLE 8 Improvements Over SEQ ID NO: 302 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 302 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 8.1 . Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 8.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 302 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 302 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 8.2.
EXAMPLE 9 Improvements Over SEQ ID NO: 338 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 338 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 9.1 . Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 9.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 338 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 338 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 9.2.
EXAMPLE 10
Improvements Over SEQ ID NO: 606 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 606 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2. Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 10.1 .
Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 10.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 606 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 606 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 10.2. Table 10.2 Table 10.2
Additionally, target bias relative to SEQ ID NO: 606 (Promiscuity FIOP) was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 606 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 10.3.
Additional reactions were run as described above but with modified conditions. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 10.4. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 10.4 and analyzed on CE as described in Example 4. Activity relative to SEQ ID NO: 606 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 606 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 10.5.
EXAMPLE 11 Improvements Over SEQ ID NO: 972 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 972 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 11.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 11.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 972 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 972 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 11.2.
Additional reactions were run as described above but with modified conditions. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 11.3. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 11.3 and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 972 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 972 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 11.4.
Additionally, target bias relative to SEQ ID NO: 972 (Promiscuity FIOP) was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 972 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 11.5. Additional target bias data were collected and calculated for each target substrate/ annealed oligo pair in Table 11.3; results (unreported) were similar to those reported below.
EXAMPLE 12 Improvements Over SEQ ID NO: 1082 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 1082 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 12.1 . Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 12.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 1082 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 1082 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 12.2.
Additionally, target bias relative to SEQ ID NO: 1082 (Promiscuity FIOP) was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 1082 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 12.3. Additional reactions were run as described above but with modified conditions. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 12.4. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 12.4 and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 1082 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 1082 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 12.5.
Additionally, target bias relative to SEQ ID NO: 1082 (Promiscuity FIOP) was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 1082 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 12.6. Additional target bias data were collected and calculated for each target substrate/ annealed oligo pair in Table 12.3; results (unreported) were similar to those reported below.
EXAMPLE 13
Improvements Over SEQ ID NO: 1218 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 1218 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2. Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 13.1.
Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 13.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 1218 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 1218 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 13.2.
Additionally, target bias relative to SEQ ID NO: 1218 (Promiscuity FIOP) was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 1218 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 13.3. EXAMPLE 14 Improvements Over SEQ ID NO: 1580 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 1580 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 14.1 . Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 14.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 1580 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 1580 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 14.2.
Additionally, target bias relative to SEQ ID NO: 1580 (Promiscuity FIOP) was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 1580 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 14.3. Additional target bias data were collected and calculated for each target substrate/ annealed oligo pair in Table 14.1 ; results (unreported) were similar to those reported below.
Additional reactions were run as described above but with modified conditions. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 14.4. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 14.4 and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 1580 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 1580 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 14.5.
Additionally, target bias relative to SEQ ID NO: 1580 (Promiscuity FIOP) was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 1580 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 14.6. Additional target bias data were collected and calculated for each target substrate/ annealed oligo pair in Table 14.1 ; results (unreported) were similar to those reported below.
Further additional reactions were run as described above but with modified conditions. The exact concentration of UR1 adapter, target substrate identity and concentration are shown in Table 14.7. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 14.7 and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 1580 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380, observed by the reaction with SEQ ID NO: 1580 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 14.8.
Additionally, target bias relative to SEQ ID NO: 1580 (Promiscuity FIOP) was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 1580 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 14.9. Additional target bias data were collected and calculated for each target substrate/ annealed oligo pair in Table 14.1 ; results (unreported) were similar to those reported below.
EXAMPLE 15
Relative Performance Measurements of Shake-Flask Purified Transposase Variants over SEQ ID NO: 972 in the Preparation of Libraries for Next Generation Sequencing Applications SEQ ID NO: 1720 was selected as the parent transposase enzyme. Variants of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptide encoded by the variants of SEQ ID NO: 1720, including SEQ ID NO: 2368, were produced in shake flask and purified as described in Example 3. Additionally, transposase variants of SEQ ID NO: 972, 1082, 1218, 1580, 1598, 1600, and 1604 were produced in shake flask and purified as described in Example 3. Transposase concentrations of select variants were measured by absorption at 280 nm and performance of the normalized enzymes was measured in the preparation of libraries for next generation sequencing as follows.
Reactions were prepared using seqWell reagents and were evaluated on an Illumina sequencer. To load the transposase enzymes the shake purified enzyme variants were diluted to 2.5uM in seqWell MTSB and combined in 1 :1 ratio with the seqWell i5 tagging reagents (premixed 2 barcode blend) and incubated at room temperature for 1 hour. The same set of samples at concentration of 2.5uM were combined with seqWell i7 oligos in 1 :1 ratio in a separate set of reactions and incubated at room temperature for one hour. The loaded i5 and i7 transposases were then combined in equal volumes and diluted to 15nM prior to library preparation. To prepare the library for sequencing 10ng of E.coli genomic DNA was mixed in equal volumes (5uL) with i5/i7 transposase mix and coding buffer. The reaction was incubated at 55°C for 15 minutes and terminated with 7.5uL of seqWell X-solution at 68°C for 10 minutes. The reaction was then purified with (1 x) MAGwise beads and eluted in 25uL of 10mM Tris pH 8.0. This was followed by 12 cycle amplification and amplification purification with MAGwise beads. The samples were evaluated using Agilent Bioanalyzer and the Illumina MiSeq assay.
When bound to appropriate adapter sequences, active transposase variants are capable of binding target sequences, cleaving the target strand, and ligating the adapter oligonucleotides to the target fragment as shown in Figure 1 .
Transposon insertion frequency is biased by the sequence of the insertion sites over the first 15 nucleotides of NGS reads putatively comprising the region of the target bound by the transposome construct. Bias plots generated from NGS data represent this bias as a function of the distance from the insertion site. Overall relative bias for each variant can be calculated as the root-mean-squared deviation (RMSD) of the observed vs. expected representation of all four nucleotides at each position as shown in Scheme 1 , shown herein. Additionally, the mean RMSD over, for example, the first 15 nucleotides (RMSD15) can be used to summarize insertion bias with a single number.
Relative target bias for each variant was calculated as the mean RMSD15 of SEQ ID NO: 972 divided by the mean RMSD15 of the transposase variant. Variants with target bias mean RMSD15 less than SEQ ID NO: 972 will have a ratio greater than 1 . The results are shown in Table 15.1 .
EXAMPLE 16 Improvements Over SEQ ID NO: 2368 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 2368 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 16.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 16.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 2368 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2375/SEQ ID NO: 2376 and SEQ ID NO: 2385/SEQ ID NO: 2386, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2375/SEQ ID NO: 2376 and SEQ ID NO: 2385/SEQ ID NO: 2386, observed by the reaction with SEQ ID NO: 2368 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 16.2.
Additionally, target bias relative to SEQ ID NO: 2368 (Promiscuity FIOP) was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2375/SEQ ID NO: 2376 vs. product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 2368 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 16.3. Additional target bias data were collected and calculated for each target substrate/ annealed oligo pair in Table 16.1 ; results (unreported) were similar to those reported below. EXAMPLE 17 Improvements Over SEQ ID NO: 2426 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 2426 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 17.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 17.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 2426 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2375/SEQ ID NO: 2376 and SEQ ID NO: 2385/SEQ ID NO: 2386 produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2375/SEQ ID NO: 2376 and SEQ ID NO: 2385/SEQ ID NO: 2386, observed by the reaction with SEQ ID NO: 2426 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 17.2.
Additionally, target bias relative to SEQ ID NO: 2426 (Promiscuity FIOP) was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2375/SEQ ID NO: 2376 vs. product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 2426 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 17.3. Additional target bias data were collected and calculated for each target substrate/ annealed oligo pair in Table 17.1 ; results (unreported) were similar to those reported below.
EXAMPLE 18 Improvements Over SEQ ID NO: 2540 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 2540 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 18.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 18.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 2540 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, observed by the reaction with SEQ ID NO: 2540 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 18.2. EXAMPLE 19 Improvements Over SEQ ID NO: 2540 in the Transposition of Double Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 2540 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 11 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 19.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 19.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 2540 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2369/SEQ ID NO: 2370, SEQ ID NO: 2371/SEQ ID NO: 2372, SEQ ID NO: 2373/SEQ ID NO: 2374, SEQ ID NO: 2375/SEQ ID NO: 2376, SEQ ID NO: 2377/SEQ ID NO: 2378, SEQ ID NO: 2379/SEQ ID NO: 2380, SEQ ID NO: 2381/SEQ ID NO: 2382, SEQ ID NO: 2383/SEQ ID NO: 2384, and SEQ ID NO: 2385/SEQ ID NO: 2386, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2369/SEQ ID NO: 2370, SEQ ID NO: 2371/SEQ ID NO: 2372, SEQ ID NO: 2373/SEQ ID NO: 2374, SEQ ID NO: 2375/SEQ ID NO: 2376, SEQ ID NO: 2377/SEQ ID NO: 2378, SEQ ID NO: 2379/SEQ ID NO: 2380, SEQ ID NO: 2381/SEQ ID NO: 2382, SEQ ID NO: 2383/SEQ ID NO: 2384, and SEQ ID NO: 2385/SEQ ID NO: 2386, observed by the reaction with SEQ ID NO: 2540 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 19.2.
Additionally, target bias relative to SEQ ID NO: 2540 (Promiscuity FIOP) was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 2540 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 19.3. Additional target bias data were collected and calculated for each target substrate/ annealed oligo pair in Table 19.1 ; results (unreported) were similar to those reported below.
EXAMPLE 20
Improvements Over SEQ ID NO: 3402 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 3402 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 20.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 20.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 3402 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2371/SEQ ID NO: 2372, SEQ ID NO: 2373/SEQ ID NO: 2374, SEQ ID NO: 2375/SEQ ID NO: 2376, SEQ ID NO: 2377/SEQ ID NO: 2378, SEQ ID NO: 2379/SEQ ID NO: 2380, SEQ ID NO: 2381/SEQ ID NO: 2382, SEQ ID NO: 2383/SEQ ID NO: 2384 and SEQ ID NO: 2385/SEQ ID NO: 2386 produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2371/SEQ ID NO: 2372, SEQ ID NO: 2373/SEQ ID NO: 2374, SEQ ID NO: 2375/SEQ ID NO: 2376, SEQ ID NO: 2377/SEQ ID NO: 2378, SEQ ID NO: 2379/SEQ ID NO: 2380, SEQ ID NO: 2381/SEQ ID NO: 2382, SEQ ID NO: 2383/SEQ ID NO: 2384 and SEQ ID NO: 2385/SEQ ID NO: 2386, observed by the reaction with SEQ ID NO: 3402 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 20.2.
Additionally, target bias relative to SEQ ID NO: 3402 (Promiscuity FIOP) was calculated as the ratio of the product peak area produced by the variant (product of the annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386 vs. product of the annealed oligos SEQ ID NO: 2379/SEQ ID NO: 2380) divided by the ratio of the product peak area observed by the reaction with SEQ ID NO: 3402 (where the control ratio may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 20.3. Additional target bias data were collected and calculated for each target substrate/ annealed oligo pair in Table 20.1 ; results (unreported) were similar to those reported below.
EXAMPLE 21 Improvements Over SEQ ID NO: 3662 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 3662 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2. Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 60 mins. After 60 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 21.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 21.1 , and analyzed on CE as described in Example 4
Activity relative to SEQ ID NO: 3662 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, observed by the reaction with SEQ ID NO: 3662 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 21.2.
Activity relative to SEQ ID NO: 3662 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 5699/SEQ ID NO: 5700, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5699/SEQ ID NO: 5700, observed by the reaction with SEQ ID NO: 3662 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 21.3.
EXAMPLE 22
Improvements Over SEQ ID NO: 3788 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 3788 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2. Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 45 mins. After 45 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 22.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 22.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 3788 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698 produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698 observed by the reaction with SEQ ID NO: 3788 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 22.2.
EXAMPLE 23
Improvements Over SEQ ID NO: 4150 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 4150 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2. Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 45 mins. After 45 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 23.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 23.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 4150 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 2385/SEQ ID NO: 2386, observed by the reaction with SEQ ID NO: 4150 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 23.2.
EXAMPLE 24 Improvements Over SEQ ID NO: 4278 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 4278 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 45 mins. After 45 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 24.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 24.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 4278 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 5695/SEQ ID NO: 5696, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5695/SEQ ID NO: 5696, observed by the reaction with SEQ ID NO: 4278 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 24.2.
EXAMPLE 25
Improvements Over SEQ ID NO: 4278 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 4278 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2. Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 45 mins. After 45 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 25.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 25.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 4278 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, observed by the reaction with SEQ ID NO: 4278 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 25.2.
EXAMPLE 26 Improvements Over SEQ ID NO: 4642 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 4642 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 45 mins. After 45 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 26.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 26.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 4642 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, observed by the reaction with SEQ ID NO: 4642 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 26.2.
EXAMPLE 27 Improvements Over SEQ ID NO: 4642 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 4642 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2. Reactions were performed in 96-well format BioRad PCR plates. 100 nL of 100% HTP lysate variants in lysis buffer (Table 27.1) were combined in 1 :1 volume ratio with 2.5 pM seqWell i5 tagging reagents and incubated at room temperature for 1 hour with mild shaking. 100 nL of the same set of samples were also combined with 2.5 pM seqWell i7 oligos in 1 :1 volume ratio in a separate set of reactions and incubated at room temperature for one hour with mild shaking. The loaded i5 and 17 transposases were then combined in equal volumes and diluted with 12.5 pL MTSB prior to library preparation. To prepare the library for sequencing 10 ng of E.coli genomic DNA was mixed in equal volumes (5 pL) with i5/i7 transposase mix and 3x coding buffer. The reactions were incubated at 55°C for 15 minutes and terminated with 7.5 pL of seqWell X-solution at 68°C for 10 minutes. The reactions were then purified with (1 x) MAGwise beads and eluted in 25 pL of 10 mM Tris pH 8.0. This was followed by 12 cycle amplification (Table 27.1), sample pooling and MAGwise bead purification. An equal volume of the samples (10 pL each) was pooled together and purified with (1 x) MAGwise beads. The resulting library pool was evaluated using NanoDrop Spectrophotometer or Qubit and the Illumina MiSeq assay was performed.
Relative target bias for each variant was calculated as the mean RMSD15 of SEQ ID NO: 4642 divided by the mean RMSD15 of the transposase variant. Variants with target bias mean RMSD15 less than SEQ ID NO: 4642 will have a ratio greater than 1 . The results are shown in Table 27.2.
EXAMPLE 28 Improvements Over SEQ ID NO: 4984 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 4984 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 45 mins. After 45 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 28.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 28.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 4984 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, observed by the reaction with SEQ ID NO: 4984 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 28.2.
EXAMPLE 29 Improvements Over SEQ ID NO: 4984 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 4984 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 45 mins. After 45 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 29.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 29.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 4984 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, observed by the reaction with SEQ ID NO: 4984 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 29.2.
EXAMPLE 30
Improvements Over SEQ ID NO: 5196 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 5196 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2. Reactions were performed in 96-well format BioRad PCR plates. 100 nL of 100% HTP lysate variants in lysis buffer (Table 30.1) were combined in 1 :1 volume ratio with 2.5 pM seqWell i5 tagging reagents and incubated at room temperature for 1 hour with mild shaking. 100 nL of the same set of samples were also combined with 2.5 pM seqWell i7 oligos in 1 :1 volume ratio in a separate set of reactions and incubated at room temperature for one hour with mild shaking. The loaded i5 and i7 transposases were then combined in equal volumes and diluted with 12.5 pL MTSB prior to library preparation. To prepare the library for sequencing 10 ng of E.coli genomic DNA was mixed in equal volumes (5 pL) with i5/i7 transposase mix and 3x coding buffer. The reactions were incubated at 55°C for 15 minutes and terminated with 7.5 pL of seqWell X-solution at 68°C for 10 minutes. The reactions were then purified with (1x) MAGwise beads and eluted in 25 pL of 10 mM Tris pH 8.0. This was followed by 12 cycle amplification (Table 30.1), sample pooling and MAGwise bead purification. An equal volume of the samples (10 pL each) was pooled together and purified with (1 x) MAGwise beads. The resulting library pool was evaluated using NanoDrop Spectrophotometer or Qubit and the Illumina MiSeq assay was performed.
Relative target bias for each variant was calculated as the mean RMSDis of SEQ ID NO: 5196 divided by the mean RMSD15 of the transposase variant. Variants with target bias mean RMSD15 less than SEQ ID NO: 5196 will have a ratio greater than 1 . The results are shown in Table 30.2.
EXAMPLE 31
Improvements Over SEQ ID NO: 5286 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 5286 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2. Reactions were performed in 96-well format BioRad PCR plates. 100 nL of 100% HTP lysate variants in lysis buffer (Table31.1) were combined in 1 :1 volume ratio with 2.5 pM seqWell i5 tagging reagents and incubated at room temperature for 1 hour with mild shaking. 100 nL of the same set of samples were also combined with 2.5 pM seqWell i7 oligos in 1 :1 volume ratio in a separate set of reactions and incubated at room temperature for one hour with mild shaking. The loaded i5 and i7 transposases were then combined in equal volumes and diluted with 12.5 pL MTSB prior to library preparation. To prepare the library for sequencing 10 ng of E.coli genomic DNA was mixed in equal volumes (5 pL) with i5/i7 transposase mix and 3x coding buffer. The reactions were incubated at 55°C for 15 minutes and terminated with 7.5 pL of seqWell X-solution at 68°C for 10 minutes. The reactions were then purified with (1 x) MAGwise beads and eluted in 25 pL of 10 mM Tris pH 8.0. This was followed by 12 cycle amplification (Table31.1), sample pooling and MAGwise bead purification. An equal volume of the samples (10 pL each) was pooled together and purified with (1 x) MAGwise beads. The resulting library pool was evaluated using NanoDrop Spectrophotometer or Qubit and the Illumina MiSeq assay was performed.
Relative target bias for each variant was calculated as the mean RMSDis of SEQ ID NO: 5286 divided by the mean RMSD15 of the transposase variant. Variants with target bias mean RMSD15 less than SEQ ID NO: 5286 will have a ratio greater than 1 . The results are shown in Table 31.2.
EXAMPLE 32 Improvements Over SEQ ID NO: 5412 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 5412 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2. Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 45 mins. After 45 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 32.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 32.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 5412 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, observed by the reaction with SEQ ID NO: 5412 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 32.2. EXAMPLE 33 Improvements Over SEQ ID NO: 5412 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 5412 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96-well format BioRad PCR plates. 100 nL of 100% HTP lysate variants in lysis buffer (Table 33.1) were combined in 1 :1 volume ratio with 2.5 pM seqWell i5 tagging reagents and incubated at room temperature for 1 hour with mild shaking. 100 nL of the same set of samples were also combined with 2.5 pM seqWell i7 oligos in 1 :1 volume ratio in a separate set of reactions and incubated at room temperature for one hour with mild shaking. The loaded i5 and i7 transposases were then combined in equal volumes and diluted with 12.5 pL MTSB prior to library preparation. To prepare the library for sequencing 10 ng of E.coli genomic DNA was mixed in equal volumes (5 pL) with i5/i7 transposase mix and 3x coding buffer. The reactions were incubated at 55°C for 15 minutes and terminated with 7.5 pL of seqWell X-solution at 68°C for 10 minutes. The reactions were then purified with (1 x) MAGwise beads and eluted in 25 pL of 10 mM Tris pH 8.0. This was followed by 12 cycle amplification (Table 33.1), sample pooling and MAGwise bead purification. An equal volume of the samples (10 pL each) was pooled together and purified with (1 x) MAGwise beads. The resulting library pool was evaluated using NanoDrop Spectrophotometer or Qubit and the Illumina MiSeq assay was performed.
Relative target bias for each variant was calculated as the mean RMSDis of SEQ ID NO: 5412 divided by the mean RMSD15 of the transposase variant. Variants with target bias mean RMSD15 less than SEQ ID NO: 5412 will have a ratio greater than 1 . The results are shown in Table 33.2.
EXAMPLE 34 Improvements Over SEQ ID NO: 5422 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences
HTP Screening for Improved Transposase Variants
SEQ ID NO: 5422 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96 or 384-well format BioRad PCR plates. Bound transposomes or synaptic complexes were prepared by incubating 67% HTP lysate with 2 - 17 pM seqWell UR1 adapter at room-temperature for 45 mins. After 45 mins incubation, annealed target substrate(s) in seqWell 3x coding buffer was added to the bound HTP transposomes to a final concentration of 0.03-0.3 pM. HTP plates were centrifuged, and samples were heated in a thermocycler at 55°C for 5-15 mins. The exact concentration of UR1 adapter, target substrate identity and concentration is shown in Table 34.1. Samples were quenched by heating at 75°C with 0.04% SDS. After heat-quench, samples were diluted with water to a final dilution as described in Table 34.1 , and analyzed on CE as described in Example 4.
Activity relative to SEQ ID NO: 5422 (Activity FIOP) was calculated as the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, produced by the variant compared with the percent product of annealed oligos SEQ ID NO: 5697/SEQ ID NO: 5698, observed by the reaction with SEQ ID NO: 5422 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 34.2. EXAMPLE 35
Improvements Over SEQ ID NO: 972 in the Transposition of Double-Stranded DNA (dsDNA) Adapters into dsDNA Target Sequences HTP Screening for Improved Transposase Variants
SEQ ID NO: 972 was selected as the parent transposase enzyme. Libraries of engineered genes were produced from the parent gene using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Example 2.
Reactions were performed in 96-well format BioRad PCR plates. 100 nL of 2.5 pM shake-flask variants prepared in Seqwell MTSB buffer were combined in 1 :1 volume ratio with 2.5 pM seqWell i5 tagging reagents and incubated at room temperature for 1 hour with mild shaking. 100 nL of the same set of samples were also combined with 2.5 pM seqWell 17 oligos in 1 :1 volume ratio in a separate set of reactions and incubated at room temperature for one hour with mild shaking. The loaded i5 and i7 transposases were then combined in equal volumes and diluted with 12.5 pL MTSB prior to library preparation. To prepare the library for sequencing 10 ng of E.coli genomic DNA was mixed in equal volumes (5 pL) with i5/i7 transposase mix and 3x coding buffer. The reactions were incubated at 55°C for 15 minutes and terminated with 7.5 pL of seqWell X-solution at 68°C for 10 minutes. The reactions were then purified with (1 x) MAGwise beads and eluted in 25 pL of 10 mM Tris pH 8.0. This was followed by 12 cycle amplification (Table 33.1), sample pooling and MAGwise bead purification. An equal volume of the samples (10 pL each) was pooled together and purified with (1 x) MAGwise beads. The resulting library pool was evaluated using NanoDrop Spectrophotometer or Qubit and the Illumina MiSeq assay was performed.
Relative target bias for each variant was calculated as the mean RMSD15 of SEQ ID NO: 972 divided by the mean RMSD15 of the transposase variant. Variants with target bias mean RMSD15 less than SEQ ID NO: 972 will have a ratio greater than 1 . The results are shown in Table 35.1 .
EXAMPLE 36 Improvements Over SEQ ID NO: 5704 in the T ransposase Activity and Library Construction for NGS
Adapter Loading of Transposases
Transposases (e.g., engineered transposases) were loaded with oligonucleotide adapters. Each oligonucleotide adapter was diluted to a concentration of 100 pM and combined in a 1 :1 ratio of top strand to bottom strand. The oligonucleotides were then incubated at 95°C for two minutes and then the temperature was reduced in a stepwise manner by 1 °C per minute until the temperature reached 25°C.
Adapter Sequences:
A14 Adapter:
A14: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG (SEQ ID NO: 5757)
20TT: /5Phos/CTGTCTCTTATACACATCT/3lnvdT/ (SEQ ID NO: 5758)
B15 Adapter:
B15: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (SEQ ID NO: 5759)
20TT: /5Phos/CTGTCTCTTATACACATCT/3lnvdT/ (SEQ ID NO: 5758)
The molar binding ratio (MBR) of an engineered transposase (e.g., an engineered Tn5 variant) was defined by the ratio of picomoles of transposase required to bind 250 picomoles of B15 adapter. For instance, 500 pmol of a Tn5 variant required to bind 250 pmol of B15 adapter is an MBR of 2.
To determine the MBR, 2.5 pL of 50 pM B15 adapter was combined with each Tn5 variant (with the respective concentration shown below in Table 36.1) at varying volumes of 0 pL, 2.4 pL, 3.6 pL, 4.8 pL, 6 pL, 7.2 pL, and 9.6 pL. The adapter was loaded into the Tn5 variant by incubating the mixture for 1 hour at 25°C on a thermocycler in which the lid was removed. The volumes of the transposomes were then raised to 16.9 pL with modified Tn5 buffer (MTSB). A sample from each tube (3.4 pL) was mixed with 19.5 pL of 10 mM Tris and were then run on a 4% agarose gel for 15 minutes. The MBR was calculated from the gel lane in which the B15 adapter was completely consumed by the Tn5 variant (i.e., a band corresponding to unbound B15 adapter was not observed).
The Tn5 variants were loaded with an adapter based on the results from the MBR assay. To ensure that all transposases were loaded with an adapter, an excess amount of an adapter was included at two-times the amount of the determined MBR concentration for each reaction. Each transposase was incubated with 2 pL of 50 pM adapter at 25°C for 1 hour. Following this incubation step, the loaded transposases were diluted to 1 pM via adding MTSB. Each transposase was loaded twice: once with an A14 adapter and again with a B15 adapter. The loaded A14 and B15 transposomes were then combined to make a 1 pM A14/B15 tagging reagent for each enzyme.
The MBR and transposase loading for each Tn5 variant is summarized in Table 36.1 . The Tn5 enzyme was obtained from a commercial source.
Transposase Activity and Performance
The activity of each transposase variant was determined using 2.5 pM B15/Tn5 variant at the selected MBR. The transposome was serially diluted 2-fold in MTSB from 1 :1 to 1 :64 volumetric dilutions. A negative control containing only MTSB without a transposome was included. Each dilution was used to linearize a pUC-19 plasmid, in which each reaction contained 100 ng of the pUC-19 plasmid, 4 pL of the transposome dilution, and 1X coding buffer. The reaction mixtures were incubated at 55°C for 15 minutes and then the reaction was stopped by adding 1 .2 pL of 0.2% SDS and incubating the reaction mixtures at 75°C for ten minutes.
After quenching the reaction, 30 pL of 10 mM Tris pH 8.0 was mixed into each reaction tube and then loaded to a 2% agarose gel (E-GEL™ Ex Agarose Gel, Invitrogen) for ten minutes. The gel lane in which all of the pUC-19 plasmid was linearized was used to calculate transposase activity. An activity score between 0-10 was assigned to each transposase variant based on the relative activity as compared to SEQ ID NO: 5704 (Table 36.3).
Exemplary NGS sequencing libraries were generated via tagmentation reactions with the transposase variants followed by amplification reactions. Each tagmentation reaction contained 1 pM of an engineered transposome (e.g., a transposome that included a Tn5 variant loaded with A14 and B15 adapters as described herein), 50 ng of human genomic DNA (NA12878), and 1X coding buffer, which were incubated at 55°C for fifteen minutes. After the incubation, the tagmentation reactions were quenched with 7.5 pL of X-Solution (transposase stop solution; seqWell, Inc) and then heated to 68°C for ten minutes.
Oligonucleotide fragments generated via tagmentation were then cleaned up with 1 ,5x solidphase reversible immobilization (SPRI) magnetic beads, which were washed twice with an 80% ethanol solution. The oligonucleotide fragments were then eluted with 12 pL of 10 mM Tris pH 8.0.
Following elution, 10 pL of the oligonucleotide fragments were combined with 6 pL of Illumina UD Adapter, and 16 pL of 2x Kapa HiFi Readymix. The oligonucleotide fragments were then amplified via PCR using the thermocycler settings summarized in Table 36.2, in which the thermocycler lid was held at a temperature of 105°C.
The resulting sequencing libraries were cleaned up via 1 .5x SPRI magnetic beads, which were washed twice with an 80% ethanol solution and then eluted in 32 pL of 10 mM Tris pH 8.0. After elution of the purified libraries from the beads, the DNA concentration was determined by electrophoresis on a
TapeStation system (Agilent), as shown in Figure 3 and Table 36.3.
The sequencing libraries were then pooled and loaded at 14pM on a MiSeq sequencing system (Illumina) using the MiSeq v2 reagents. The data was demultiplexed and aligned using Picard tools. Insertion bias was measured by calculating the RMSE of the first 25 bases of the insert using the following equation:
The predicted values were 29.5% for A and T and 20.5% for C and G based on the average base composition of the human genome. For each Tn5 variant, the RMSE for nucleobase positions 1 -20 were added, and the calculated insertion bias values for the transposases were compared (Figures 4A-4D and Table 36.3). These data highlight Tn5 transposase variants that have improved function over the SEQ ID NO: 5704. Some variants, including the one set forth in SEQ ID NO: 5738, exhibited a lower insertion bias as shown in Table 36.3.
EXAMPLE 37
Loading of Transposase Reagent with Unique Dual-Indexed Adapters
A method was developed for loading transposases, i.e. , engineered transposases, with unique dual-indexed adapters.
Sequences Used
/7 Index Adapters (Top Strand Sequence Shown)
Identifier: i7-1437
Sequence:
CAAGCAGAAGACGGCATACGAGATTGGACTTGACGTCTCGTGGGCTCGGAGATGTGTATAAGAGAC AG (SEQ ID NO: 5760)
Identifier: i7-1610
CAAGCAGAAGACGGCATACGAGATGGAAGAGATAGTCTCGTGGGCTCGGAGATGTGTATAA GAGACAG (SEQ ID NO: 5761 )
Identifier: i7-0790
CAAGCAGAAGACGGCATACGAGATTTCTTCGCGGGTCTCGTGGGCTCGGAGATGTGTATAA GAGACAG (SEQ ID NO: 5762)
Identifier: i7-0822
CAAGCAGAAGACGGCATACGAGATTCCGAGTAGGGTCTCGTGGGCTCGGAGATGTGTATAA GAGACAG (SEQ ID NO: 5763)
Identifier: i7-1872
CAAGCAGAAGACGGCATACGAGATGTGATACGAAGTCTCGTGGGCTCGGAGATGTGTATAA GAGACAG (SEQ ID NO: 5764)
Identifier: i7-1666
CAAGCAGAAGACGGCATACGAGATGGATGGAGGAGTCTCGTGGGCTCGGAGATGTGTATAA GAGACAG (SEQ ID NO: 5765)
Identifier: i7-0474
CAAGCAGAAGACGGCATACGAGATGCGATGTTATGTCTCGTGGGCTCGGAGATGTGTATAA GAGACAG (SEQ ID NO: 5766)
Identifier: 17-1201
CAAGCAGAAGACGGCATACGAGATAACGCATATCGTCTCGTGGGCTCGGAGATGTGTATAA GAGACAG (SEQ ID NO: 5767) i5 Index Adapters (Top Strand Sequence Shown)
Identifier: i5-1714
AATGATACGGCGACCACCGAGATCTACACGTAACACAGATCGTCGGCAGCGTCAGATGTGT
ATAAGAGACAG (SEQ ID NO: 5768)
Identifier: i5-0744
AATGATACGGCGACCACCGAGATCTACACTAAGTTGTGGTCGTCGGCAGCGTCAGATGTGTA
TAAGAGACAG (SEQ ID NO: 5769)
Identifier: i5-1180
AATGATACGGCGACCACCGAGATCTACACCACCTACCTCTCGTCGGCAGCGTCAGATGTGTA
TAAGAGACAG (SEQ ID NO: 5770)
Identifier: i5-1347
AATGATACGGCGACCACCGAGATCTACACATATCAGTCCTCGTCGGCAGCGTCAGATGTGTA
TAAGAGACAG (SEQ ID NO: 5771 )
Identifier: i5-0995
AATGATACGGCGACCACCGAGATCTACACGGTTAAGTAGTCGTCGGCAGCGTCAGATGTGT
ATAAGAGACAG (SEQ ID NO: 5772)
Identifier: i5-0500
AATGATACGGCGACCACCGAGATCTACACTGGTGGATATTCGTCGGCAGCGTCAGATGTGTA
TAAGAGACAG (SEQ ID NO: 5773)
Identifier: 15-1482
AATGATACGGCGACCACCGAGATCTACACTAGCTGGCACTCGTCGGCAGCGTCAGATGTGT
ATAAGAGACAG (SEQ ID NO: 5774)
Identifier: i5-0732
AATGATACGGCGACCACCGAGATCTACACTTACCGTTGGTCGTCGGCAGCGTCAGATGTGTA
TAAGAGACAG (SEQ ID NO: 5775)
Identifier: 20TT
/5Phos/CTGTCTCTTATACACATCTG (SEQ ID NO: 5776)
1 Adapter Annealing
Mix top strand oligo and mosaic end bottom strand oligo (20TT) as equimolar oligo dilutions using oligo annealing buffer
2 min at 95 °C
Thermocycler Protocol (steps (i) through (iii))
(i) 2 min at 95 °C
(ii) 1 min at 94 °C
(iii) 1 min ramp down: -1 °C each cycle hold 20 °C
2 Transposase + Adapter complex formation
Mix equimolar Adapter and transposase
Incubate 1 hr at RT 3 Mix in Modified Transposase Storage Buffer (MTSB) to bring synaptic complex to < 5pM
4 Repeat above for both the i7 and i5 barcodes
5 Mix i7 and i5 barcode equally and dilute to working concentration in MTSB
EXAMPLE 38 Activity Assay for Loaded Transposase
Engineered transposases were assayed for relative activity by measuring the molar amount of adapter-loaded transposase required to convert specific molar amount of closed circular plasmid DNA standard to a singly cut linear DNA molecule. Specifically, 2.5 pmol of each adapter-loaded transposase was used to convert a series of pUC19 dilutions centered at 200 ng to linear form after incubation at 55°C for 15 minutes in 1x Coding Buffer. The tagmentation reaction was stopped by adding SDS and incubating at 75C for 10 minutes. Samples were diluted and loaded in a 2% E-Gel for 10 minutes. In the following example, 2.5 pmol of engineered transposase completely converted 100 ng of pUC-19 to the linear form (performed with SequenceK (SEQ ID NO: 5499/5500 for nt/aa); see Figure 5A), while 2.5 pmol of commercially available transposase was only able to completely convert all the 50 ng input of pUC-19 to linear form (performed with commercial hyperactive Tn5; see Figure 5B). The fold improvement in this case was 2x (100 ng / 50ng = 2). In particular, the linear form is shown as the band and/or smear that starts at ~3000bp in Figure 5A and Figure 5B.
EXAMPLE 39. Unique Dual-Indexed Transposition for next-generation sequencing (NGS) Library Generation
The engineered transposases were used to generate an exemplary NGS library using the protocol described below. Reactions may be performed, for example, in a 96-well plate.
1 . Tagging Reaction (in each reaction vessel, e.g., each well of a 96-well plate)
DNA 8 pL (50-200 ng total) (purified human genomic DNA from Coriell NA12878) i7+i5 transposase 8 pL 3x Coding Buff 8 pL TOTAL: 24 pL
Tag Thermocycler Protocol: 15min at 55 °C Hold at 25 °C
2. Stop Reaction
Mix in 12 pL X-Solution (transposase stop solution) (seqWell, Inc)
Stop Reaction Thermocycler Protocol:
10 min at 68 °C
Hold at 25 °C
3. Pool samples
Pipette 32 pL from each stopped reaction
Samples are pooled by row of a 96-well plate into 8 wells 4. SPRI Cleanup
0.75x - 1x solid-phase reversible immobilization (SPRI) cleanup (magnetic bead cleanup) based on desired library size
2 x 80% EtOH Wash
Elute in 48 pL 10mM Tris
5. Library Amplification
Library - 46 pL total volume from elution above
Add 4 pL 50pM Library Primer Mix
Add 50 pL 2x Kapa HiFi
6. Post Amplification SPRI Cleanup
1x SPRI cleanup
2x 80% EtOH Wash
Elute in 30 pL 10 mM Tris
The exemplary NGS library was analyzed using Agilent 2100 Bioanalyzer automated electrophoresis to analyze library fragment size. Results are shown in Figure 6. Similar to results shown above in Example 38, transposition using the engineered transposase completely converted input DNA sample into linear form, regardless of input amount (50-200 ng). The resulting electopherogram describes the composition of the library.
EXAMPLE 40
Comparison of Sequencing Performance of Different Engineered Transposases in Bacterial WGS
Engineered transposases were compared based on their performance in generating libraries for bacterial whole genome sequencing. Transposase activity of the different engineered transposases were determined according to the protocol in Example 38. Loaded transposase library reagents were prepared according to the protocol described in Example 37 using a distinct combination of Illumina-compatible i5/p5 and i7/p7 barcoded adapters for transposase loading.
Transposition reactions were performed, and Illumina-compatible libraries were generated according to the protocol described in Example 39 from genomic DNA isolates of three microbial species: Staphylococcus epidermidis, Bacillus subtilis, and Pseudomonas aeruginosa. These three microbial species were selected to represent a range of GC-containing genomic sequence content.
The resulting libraries were pooled and sequenced together on an Illumina NextSeq2000 instrument, and the resulting data were demultiplexed and analyzed by reference-based mapping and analysis using bwa-mem and Picard. Resulting summary statistics were collected for each transposase (e.g., engineered transposase or hyperactive Tn5 control) and are reported below.
Metrics Collected:
Norm GC (slope) - The slope of normalized coverage by GC bin in 1000 bp windows of genomic sequence FoldSO Penalty - The required excess of sequencing depth that would be required to bring 80% of covered bases to the median level of coverage
CV of Coverage - The standard deviation of coverage divided by the mean of coverage
% Duplication - The percentage of mapped reads having identical starting and ending genomic coordinates
Unique Insert Positions - The percentage of reads having a unique insert starting or ending genomic coordinate
Performance of Engineered Transposases:
The metrics selected for analysis enable performance characterization of each variant relative to each other and to hyperactive Tn5 control (Tn5).
To obtain a global representation of each transposases’ effect on sequence integrity, transposase-induced GC bias was examined across three bacterial genomes representing a range of GC- containing genomic sequence {Staphylococcus epidermidis - 32% GC, Bacillus subtilis - 44% GC and Pseudomonas aeruginosa - 66% GC) using Picard GC bias plots. Low GC bias would produce a normalized coverage slope of one, or a horizontal line over windows of GC content. Alternatively, high GC bias would produce a normalized coverage slope greater or less than one, over windows of GC content.
Quantified performance of the engineered transposases in Staphylococcus epidermidis, Bacillus subtilis, and Pseudomonas aeruginosa are provided below in Tables 40.1-40.3.
Results from libraries generated from the low GC Staphylococcus epidermidis (32% GC) genome (Table 40.1) indicated that SequenceE and SequenceH have the least bias for high AT sequences. SequenceC and SequenceJ performed best from libraries generated from the more balanced Bacillus subtilis (44% GC) genome (Table 40.2), and SequenceM performed best from libraries generated from the high GC Pseudomonas aeruginosa (66% GC) (Table 40.3).
The fold-80 penalty measures the amount of excess sequencing required to bring 80% of covered bases to the median level of coverage. A completely unbiased system would return a score of one. For all 3 bacterial genomes (Tables 40.1-40.3), all variants with the exception of SequenceA performed better than commercial hyperactive Tn5, indicating generally higher complexity and more uniform coverage libraries from these variants.
Another uniformity of coverage metric, CV of coverage, confirmed the fold-80 penalty results. Here, all variants with the exception of SequenceA for all bacterial genomes (Tables 40.1-40.3), and SequenceM, SequenceH, Sequencel, and SequenceJ for libraries generated from the high GC Pseudomonas aeruginosa (66% GC) genome performed better than hyperactive Tn5.
Library complexity can be measured by calculating the percentage of reads having a unique insert starting or ending genomic coordinate.
EXAMPLE 41
Comparison of Sequencing Performance of Different Engineered Transposases in Human WGS
Engineered transposases were compared based on their performance in generating libraries for human whole genome sequencing. Transposase activity of the different engineered transposases was determined according to the protocol in Example 38. Loaded transposase library reagents were prepared according to the protocol described in Example 37 using a distinct combination of Illumina-compatible i5/p5 and i7/p7 barcoded adapters for transposase loading.
Transposition reactions were performed in triplicate for each of the engineered transposases and hyperactive Tn5 control, and Illumina-compatible libraries were generated in the manner described from 100 ng of human NA12878 genomic DNA for each condition tested. Human DNA was selected to represent a high complexity genomic content to enable analysis of transposition insertion sites and to measure the impact on coverage uniformity and library complexity metrics.
The resulting libraries were pooled and sequenced together on an Illumina NovaSeq X instrument, and the resulting data were demultiplexed and analyzed by reference-based mapping and analysis. Resulting summary statistics were collected for each transposase (e.g., engineered transposase or hyperactive Tn5 control) and are reported below.
Metrics Collected after down sampling to 620 million reads per sample:
Library size - The estimated library size after de-duplication.
Picard: GC Coverage Bias - The relative proportions of guanine (G) and cytosine (C) nucleotides in a sample
Picard WGS Coverage - The coverage in bases of the genome territory, after all filters are applied.
Per base sequence content - Heatmap of the per base sequence content at the transposition insertion site
Transposase Performance:
The estimated sizes of the NGS libraries prepared by the different commercial transposases and engineered transposases are shown in Figure 7. The GC coverage bias of the NGS libraries prepared by the different commercial transposases and engineered transposases are shown in Figure 8.
The WGS coverage of the NGS libraries prepared by the different commercial transposases and engineered transposases are shown in Figure 9.
The per base sequence content of the transposition sites of the different commercial transposases and engineered transposases are shown in Figure 10.
Each nucleotide (A, T, G, or C) in Figure 10 is represented by a single line trace, representing the percentage of reads having that nucleotide at the corresponding position relative to the read start site (transposition site) of the DNA molecules in the sequenced library. Engineered Transposases SequenceK and SequenceH showed improvements in the uniform representation of nucleotides at positions where alternate transposases (Illumina DNA Prep and commercial hyperactive Tn5 Transposase) have specific nucleotides that are overrepresented. For example, commercial hyperactive Tn5 shows a higher bias and percentage of reads with “C” at position 9 of sequenced reads, than either enzyme SequenceK or SequenceH.
EXAMPLE 42
Evaluation of Engineered Transposases in ExpressPlex for Bacterial WGS
To compare the performance of engineered transposases in alternate buffer systems that are less optimal for transposase activity, engineered transposases were tested in the context of the ExpressPlex assay (seqWell, Inc.). Experimental tagging reagents were prepared by binding full-length sequencing adapters to transposase proteins (engineered transposases and control hyperactive Tn5 transposase). Then, libraries were prepared from different bacterial genomic DNA isolates using these tagging reagents, as follows:
1 . 4 pL of DNA and 4 pL of the tagging reagent were added to 8 pL of standard Ready Reaction reagent (seqWell, Inc.) in 96-well PCR plates.
2. Next, the 96-well plate was sealed and placed in a thermal cycler. The following thermal cycling program (Table 42.1) was used to tag the DNA and amplify the libraries:
3. After completion of the thermal cycling program, 10 pL of each reaction was pooled together in a single tube and purified with 0.75X MAGwise paramagnetic beads (seqWell, Inc.).
4. After elution of the purified library from the beads, the DNA concentration was determined by electrophoresis on a TapeStation system (Agilent).
5. The library was denatured and loaded onto a MiSeq sequencing system (Illumina), and run in the 2 x 151 base read configuration.
6. The output sequencing data were demultiplexed and aligned to their respective DNA reference sequences, and assessed for uniformity of coverage.
GO bias was evaluated for NGS libraries prepared by ExpressPlex using hyperactive Tn5 transposase and the SequenceH engineered transposase from Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. Results are shown in Figure 11.
EXAMPLE 43
Evaluation of Mutant Transposase Variants in ExpressPlex for Plasmid Sequencing
To compare the performance of transposase variants in alternate buffer systems that are less optimal for transposase activity, engineered transposases were evaluated in the context of the ExpressPlex assay (seqWell, Inc.). Experimental tagging reagents were prepared by binding full-length sequencing adapters to transposase proteins (engineered transposases and control hyperactive Tn5 transposase). Then, libraries were prepared from purified plasmid samples and PCR products derived from lambda DNA, using these tagging reagents, as follows:
1 . 4 pL of DNA and 4 pL of the tagging reagent were added to 8 pL of standard Ready Reaction reagent (seqWell, Inc.) in 96-well PCR plates.
2. Next, the 96-well plate was sealed and placed in a thermal cycler. The following thermal cycling program (Table 43.1) was used to tag the DNA and amplify the libraries:
3. After completion of the thermal cycling program, 10 pL of each reaction was pooled together in a single tube and purified with 0.75X MAGwise paramagnetic beads (seqWell, Inc.).
4. After elution of the purified library from the beads, the DNA concentration was determined by electrophoresis on a TapeStation system (Agilent).
5. The library was denatured and loaded onto a MiSeq sequencing system (Illumina), and run in the 2 x 151 base read configuration.
6. The output sequencing data were demultiplexed and aligned to their respective DNA reference sequences, and assessed for uniformity of coverage and GO bias.
Result of the uniformity of coverage and the GC bias analysis in raw data and analyzed as the coefficient of variation (CV) is found in Figure 12 (Lambda amplicon 18) and Figure 13 (pUC19). As measured by CV, the variability in coverage produced by engineered transposases SequenceE, SequenceH, and SequenceK produced superior uniformity of coverage (lower CV) versus commercial hyperactive Tn5 transposase. Because the ExpressPlex reaction conditions were optimized for both transposase and PCR polymerase activity, these results also demonstrated the improved robustness of engineered transposases SequenceE, SequenceH, and SequenceK when tested under altered reaction conditions. OTHER EMBODIMENTS
While the invention has been described with reference to the specific embodiments, various changes can be made and equivalents can be substituted to adapt to a particular situation, material, composition of matter, process, process step or steps, thereby achieving benefits of the invention without departing from the scope of what is claimed.
For all purposes in the United States of America, each and every publication and patent document cited in this disclosure is incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an indication that any such document is pertinent prior art, nor does it constitute an admission as to its contents or date.

Claims

WHAT IS CLAIMED IS:
1 . An engineered transposase comprising a polypeptide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, or a functional fragment thereof, wherein the engineered transposase comprises at least one substitution or substitution set in its polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704.
2. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 10, 13, 24, 35, 39, 44, 46, 51 , 54, 55, 62/361 , 72, 75, 82, 89, 92, 107, 128, 137, 164, 167, 177/359, 191 , 192, 193, 194, 201 , 217, 281 , 289, 291 , 291 /340, 305, 309, 310, 317, 331 , 337, 340, 346, 352, 353, 357, 358, 359, 362, 371 , 396, 414/435, 415, 428, 437, 443, 447, and 448, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2.
3. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 4, 5, 6, 8, 10, 10/317, 10/317/358, 10/317/359, 10/346, 10/346/359, 10/358, 10/359, 12, 12/373, 13, 15, 16, 18, 19, 26, 30, 30/154, 31 , 34, 35, 37, 38, 68, 70, 73, 74, 93, 106, 118, 126, 134, 135, 143, 146, 154/453, 163, 182, 207/286, 208, 226, 237, 246, 247, 259, 262, 263, 264, 280, 282, 286, 287, 288, 289, 291 , 292, 293, 298, 305, 309, 310, 313, 317, 317/346, 317/359, 317/445, 320, 346, 351 , 355, 356, 358, 359, 360, 368, 369, 410, 411 , 422, 426, 427, 428, 446, 448, 453, and 454, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4.
4. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 18/73/74/259, 18/73/286/289/411 ,
18/74/118/259/286/289/411 , 18/74/259, 18/74/259/286/289, 18/74/259/286/411 , 18/118/259, 18/259/286/289/411 , 18/259/289/411 , 18/289/411 , 73/74/259/286/289, 73/74/259/289, 73/259/286, 74/259, 74/259/289, 74/286/289/411 , 259, 259/286/289, and 289, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 156.
5. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 16/19/355/427,
16/37/146/286/287/289/310/313/355, 16/146/226/287/289/355, 19/37/146/286/287/310/355, 19/135/286/287/289/355, 19/182/355, 37/70/226/310/313/355, 37/74/310/355/427,
37/135/146/182/226/286/287/355/368, 37/146/226/289/427, 37/146/287/289/427, 38, 41 , 42, 43, 44, 45, 47, 48, 50, 53, 54, 55, 70/74/226/286/287/289/368/427, 70/74/355/368, 70/135/146/150/182/286/289/313/427, 74/310/313/355, 74/310/355, 74/355/427, 101 , 103, 1 17, 135/226/286/289/355/368, 135/355, 146, 146/150/287/289/310/355, 146/182/355, 146/226/286/355, 146/286/287/289/355/427, 146/289, 146/427, 150/226/310/313, 182/226/355, 182/355, 244, 247, 249, 251 , 287/355/427, 300, 317/41 1 , 355, 393, 397, 398, 399, 401 , 405, 407, 408, 409, 41 1 , 413, 426, 429, 431 , 439, 444, 446, 450, 453, 454, 457, 459, 460, 464, 466, 467, 469, 470, 471 , 472, 474, 475, 476, and 478, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 302.
6. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 16, 16/313, 16/409/41 1 /427,
37/150/408, 37/313/317/408/41 1 , 37/317/405/408, 37/317/405/426, 37/405/409/427, 150, 150/313/405/41 1 , 150/405/406, 150/408, 150/408/41 1 , 226/405, 317/460, and 408/426, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 338.
7. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 12, 13, 18, 19, 22, 26, 31 /262, 34, 60, 79, 80, 81 , 82, 92, 106, 107, 1 13, 1 15, 132, 134, 136, 140, 143, 152, 160, 163, 167, 168, 175, 179, 183,
195, 199, 201 , 202, 215, 216, 218, 220, 223, 226, 233, 235, 236, 237, 243, 255, 259, 261 , 262, 266, 268, 270, 273, 282, 290, 292, 293, 309, 319, 320, 326, 343, 346, 365, 370, 374, 380, 382, 386, 389, 390, 408, 412, 414, 415, 424, and 442, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 606.
8. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 2, 12, 13, 15, 16, 18, 19, 22, 26, 30, 31/262, 34, 51 , 60, 69, 79, 82, 84, 87, 88, 106, 107, 1 13, 1 14, 1 15, 128, 132, 134, 135, 136, 140, 143,
146, 150, 152, 160, 163, 167, 168, 175, 179, 183, 195, 198, 199, 201 , 215, 217, 218, 219, 220, 223, 224,
226, 227, 231 , 233, 235, 236, 243, 254, 255, 259, 261 , 266, 267, 268, 270, 272, 273, 275, 277, 279, 282,
287, 289, 290, 291 , 292, 293, 304, 307, 309, 319, 320, 343, 344, 346, 355, 365, 370, 373, 380, 382, 386,
389, 390, 408, 412, 414, 415, 418, 423, 424, and 468, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 606.
9. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 44/45/47/408/453, 44/45/47/453, 44/45/47/453/454, 44/45/103/453/457, 44/45/453/454/457, 44/47/103/393/408/453/454,
44/47/103/453/454/457, 44/47/393/408/453/457, 44/47/393/453/457, 44/103/408/453/454, 45/47/103/393/453/454/457, 45/47/393/453/454, 45/47/408/423/453/457, 45/47/453, 45/103/393/453/454, 45/453/454, 47/103/408/453/454, 47/393/408/453/454/457, 47/393/453/454, 47/408/453/457, 53/54/244/450, 103/393/408/453/457, 244/450, 408/453/454, 446/450/464, and 453/457, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 606.
10. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 38/45/53, 38/45/53/54/476, 38/45/53/244, 38/45/53/470/472, 38/45/54/244, 38/45/54/470/474/478, 38/45/472/476/478, 38/45/476, 38/53/54, 38/53/54/244, 38/53/54/244/472/478, 38/53/472, 38/54/244/470/474/476/478, 38/244/470/472/474/478, 38/478, 45/53/54/244/470/474/476/478, 45/53/54/470/474/476, 45/53/54/472/478, 45/54/244/474/478, 53, 53/54/470/476/478, 53/244/474/478, 54, 54/244, 54/244/472/476, 54/472, 244, and 474/478, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972.
1 1 . The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 22/38/45/107/259/317/412, 22/38/45/259/317/412, 22/107, 22/107/317/412, 22/134, 22/134/220, 22/134/220/317, 22/134/317/412, 22/317, 38, 38/45/81/143/244, 38/53/243, 38/81 , 38/81/244, 38/107/134/220, 38/132, 38/220/317, 45/81 , 45/81/243, 45/81/243/343, 45/143/243/270/343, 45/244/270, 53/1 15/163/243/270/343, 53/143/243/270/343, 53/270, 64/134/220/317/412, 81/132/243, 107, 107/134/317, 1 15/244/270, 134/259, 143/243/270, 243, 243/270, 244, 244/270, 259, 270, and 317, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972.
12. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 22/38/45/107/259/317/412, 22/38/45/259/317/412, 22/38/64/134/220, 22/45/220/259, 22/64/107/259/317/412, 22/64/134/220, 22/107, 22/134, 22/134/317, 22/134/317/412, 22/220, 22/220/412/415, 22/259/317, 22/259/317/412, 22/317, 38, 38/45/81 /143/244, 38/45/143/270, 38/53/243, 38/81 , 38/81/143/243/270, 38/81/183/243/270, 38/81/243, 38/81/244, 38/107/134/220, 38/132, 38/220/317, 38/270/343, 45/81 , 45/81/132/243/270, 45/81/243, 45/143/243/270/343, 45/244/270, 53/143/243/270/343, 53/244/270, 53/270, 64, 64/107/134, 64/107/317, 64/134, 64/134/220/317/412, 81/132, 81/132/243, 107, 107/134/317, 107/412, 1 15/244/270, 143/243/270, 220/259, 243, 243/270, 244/270, 255, 259, and 270, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972.
13. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 143, 143/175/272/273/415, 152/168/216/237, 152/168/423, 160, 160/201/272, 160/201/273/415, 160/226, 160/244/272/273/415, 160/415, 168, 168/187/216/255/423, 168/216/237, 168/255/423, 168/290/423, 187, 187/423, 199/201/226, 199/201/415, 201 , 216, 216/423, 226/244/415, 226/415, 237, 237/290, 244, 255, 272, 290, 290/423, 415, and 423, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1082.
14. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 143, 143/160/226/415, 143/175/272/273/415, 152/168/216/237, 152/168/423, 160, 160/201/272, 160/201/273/415, 160/226, 160/244/272/273/415, 160/415, 168, 168/187/216/255/423, 168/216/237, 168/216/237/255/343, 168/255/423, 168/290/423, 187, 199/201/226, 199/201/415, 201 , 216, 216/423, 226/244/415, 226/415, 237, 237/290, 244, 255, 255/343, 272, 290, 290/423, 415, and 423, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1082.
15. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 72, 90, 91 , 92, 93, 95, 122, 129, 141 , 142, 143, 144, 145, 150, 151 , 151/155, 152, 154, 155, 157, 160, 161 , 161/313, 164, 165, 168, 180, 181 , 184, 193, 197, 204, 205, 205/472, 206, 299, and 320, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1082.
16. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 90, 92, 122, 129, 141 , 143, 144, 145, 150, 151 , 151/155, 152, 154, 155, 155/265, 157, 160, 161 , 164, 165, 168, 180, 181 , 184, 193, 197, 204, 205, 205/472, 206, 299, and 320, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1082.
17. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 129, 129/160, 129/160/164, 129/160/184, 129/164, 129/164/184, 129/205, 146/152/244, 150/155/226, 150/155/244, 152, 154, 154/160/184, 154/164, 154/205, 155, 160, 160/164, 160/164/184, 160/184, 164, 164/204, 164/205, 204, 205, 226, 226/244, and 244, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1218.
18. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 129, 129/143, 129/154/164, 129/154/164/205, 129/154/205, 129/160, 129/160/164, 129/160/205, 129/164/205, 143, 143/150/155/226/244, 143/150/155/244, 143/150/226, 143/150/226/244/343, 143/150/226/343, 143/150/244, 143/150/343, 143/152, 143/152/226, 143/152/226/244, 143/152/226/343, 143/152/343, 143/155, 143/155/226/244, 143/155/226/244/343, 143/155/343, 143/226, 143/226/244/343, 143/226/343, 143/244, 143/244/343, 143/343, 146/152/244, 150/155/226, 150/155/244, 150/155/244/343, 150/226/244/343, 152, 152/174/244/343, 152/226, 152/226/343, 152/343, 154, 154/160/184, 154/164, 154/164/204, 154/164/204/205, 154/205, 155, 160, 160/164, 160/164/184, 164, 205, 226, 226/244, and 244, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1218.
19. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 98/129/157/168, 129/157/168, 151 , 151/152/255, 151/152/273, 151/255, 151/343, 157, 157/164, 157/164/168, 157/168, 164, 164/255, 168, 183, 255, 255/343, and 343, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
20. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 129, 129/157, 129/157/168, 129/164, 129/164/168/183, 129/164/183, 143, 143/151 , 143/151/152, 143/151/164, 143/164, 143/164/255, 143/164/343, 143/255, 143/343, 151 , 151/152, 151/152/164/343, 151/152/343, 151/255, 151/255/343, 151/343, 152, 152/255, 157, 157/164/168, 157/168, 164, 164/168, 164/255, 164/447, 168, 183, 255, 255/343, 298/343, and 343, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
21 . The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 69, 69/164, 72, 73, 91 , 92/164, 95, 96,
97, 98, 110, 112, 113, 121 , 121/164, 122, 122/164, 124, 126, 127, 139, 140, 141 , 144, 144/164, 145, 146, 147, 148, 150, 150/164, 151 , 151/164, 153, 153/164, 155, 156, 157, 159, 160, 161 , 163, 164, 164/172, 164/173, 164/182, 164/188, 164/191 , 164/192, 164/195, 164/197, 164/199, 164/202, 164/205/249, 164/209, 165, 166, 167, 169, 170, 171 , 172, 173, 177, 184, 187, 188, 190, 191 , 192, 193, 194, 195, 197,
197/266, 198, 199, 200, 202, 203, 204, 205, 208, 209, 210, 321 , 324, 331 , and 349, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
22. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 69, 69/164, 72, 73, 91 , 92/164, 95, 97,
98, 121/164, 126, 127, 139, 140, 142, 144, 144/164, 145, 146, 146/164, 148, 150, 150/164, 151 , 153, 153/164, 155, 156, 157, 160, 163, 164/173, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/205/249, 164/209, 165, 166, 167, 169, 171 , 172, 173, 177, 182, 187, 188, 190, 191 , 192, 194, 195, 197/266, 198, 199, 200, 202, 203, 204, 205, 208, 209, 210, 324, 331 , and 349, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
23. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 73, 91 , 93, 95, 97, 98, 110, 112, 122, 122/164, 124, 127, 139, 144, 145, 146, 146/164, 147, 148, 150, 150/164, 151 , 151/164, 153, 153/164, 155, 156, 157, 159, 160, 161 , 162, 163, 164, 164/182, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/205/249, 164/209, 165, 166, 167, 169, 171 , 172, 177, 184, 187, 188, 190, 191 , 192, 193, 195, 197/266, 198, 199, 200, 204, 205, 206, 207, 208, 209, 210, and 324, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
24. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 69, 73, 91 , 127, 140, 144, 145, 146, 146/164, 150, 150/164, 151 , 153, 155, 156, 157, 160, 163, 164, 164/172, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/209, 165, 167, 169, 171 , 172, 182, 183, 188, 191 , 192, 193, 195, 196, 198, 199, 200, 204, 205, 208, 210, and 324, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
25. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 38/143/168/220/290/317/423,
38/152/154/164/168/220/290/317/423, 38/152/168/220/290/317/343/423, 38/152/168/220/290/317/423, 38/154/164/168/220/290/317/423, 38/168/220/290/317/423, and 38/220/317, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972.
26. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 73/143/169/191/193, 73/156, 73/156/192/435, 73/156/210, 73/163, 73/163/210, 73/192/193, 127, 127/160/172/198, 127/172, 127/198, 146, 146/151/188, 146/324, 151/171/324, 151/324, 156/169, 156/192, 157/171 , 163, 163/169, 163/169/191/192/193, 165/171/324, 165/188/324, 165/324, 169, 171/188/324, 171/324, 172, 188, 192, 192/193, 198, 210, and 324 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2368.
27. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 73, 73/163/191/192/193, 127/160/172, 127/160/172/198, 144, 160, 160/172, 163/191/192, and 192 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2368.
28. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 8, 13, 17, 18, 20/146/157/167, 26, 34/38, 38/39/45, 67, 70, 73, 73/150/155/169, 73/150/171/192, 73/155, 73/155/169/199, 73/155/171 , 73/192, 73/199, 78, 82, 84, 86, 86/384, 89, 90, 91 , 95, 102, 110, 111 , 113, 114, 115, 122/127, 126/127, 127/131/165, 127/133, 143, 143/146, 143/146/153, 143/146/157, 143/146/167, 143/146/167/188, 143/146/188, 143/153/167/188/246, 143/156/157/167/195, 143/156/188, 143/188, 143/188/195, 143/195, 144, 145, 145/151 , 146, 146/152, 146/153/157, 146/153/157/167, 146/153/167, 146/157, 146/157/167, 146/167, 150, 150/155/169, 150/155/169/192, 150/155/192, 150/169, 150/192, 150/199, 151/152/154, 152/154, 152/154/155/160, 153/157/167, 155/171 , 155/192, 156/270, 160/371 , 167/188, 168/172, 169, 184, 190, 192/198, 198, 206, 208, 209, 210, 214/220, 215/220, 216/220, 217/220, 219/220, 220, 220/222, 220/225, 220/226, 231 , 235, 242, 257, 259, 261 , 262, 267, 268, 269, 270, 272, 274, 275, 276, 277, 278, 282, 283/290, 284/290, 287/290, 288/290, 289/290, 290, 290/291 , 290/292, 290/293, 290/294, 304, 305, 305/373, 306, 307, 309, 317, 329, 333, 334, 339, 341 , 344, 345, 346, 352, 353, 355, 358, 366, 368, 370, 372, 373, 375, 376, 378, 379, 380, 381 , 382, 383, 384, 385, 386/393, 387/393, 389/393, 390/393, 391/393, 392/393, 393/394, 414, 415, 420/423, 422/423, 423, 423/425, 432, 440, 441 , 443, 469, and 472 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2426.
29. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 8, 18, 25, 29, 30, 67, 73,
73/150/155/169, 73/150/155/171 , 73/150/171 /192, 73/171 , 73/192, 73/199, 74, 82, 86, 86/384, 113, 115, 118, 127/128, 143, 150, 150/155/169, 150/155/169/192, 150/155/171 , 150/155/171/199, 150/155/192, 150/155/199, 150/192, 150/199, 155/192, 168/171/172, 195/198, 197/198, 198/201 , 198/203, 198/205, 235, 252, 256, 257, 262, 263, 267/343, 268, 269, 270, 272, 273, 276, 277, 288/290, 289/290, 290, 290/291 , 290/292, 290/294, 298, 304, 305, 307, 333, 346, 355, 366, 370, 386/393, 389/393, 393/395, 440, and 472 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2426.
30. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 66, 67, 70, 101 , 102, 103, 104, 106,
107, 108, 113, 114, 115, 117, 118, 143, 152, 154, 235, 236/251 , 237, 238, 242, 243, 246, 247, 249, 250, 251 , 256/352, 257, 259, 285, 286, 287, 288, 289, 290, 292, 293, 294, 295, 296, 322, 323, 326, 329, 330, 333, 339, 340, 343, 344, 345, and 352. and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2540.
31 . The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 18, 18/113/270, 18/113/270/329/370, 18/113/270/370/384, 18/113/270/370/412, 18/113/329/384, 18/259/370/384, 18/270, 18/270/370,
18/270/370/384, 18/370, 18/370/384, 73, 73/115, 73/115/184/333, 73/115/184/341 , 73/115/256, 73/184/256, 73/184/273/333, 73/184/333, 73/256, 73/256/375, 73/273, 73/273/333/375, 73/273/375, 73/333, 73/333/375, 73/375, 73/379, 77/233/238/376, 77/233/334/376, 77/262/376, 77/276, 77/334/376, 82, 82/127, 82/127/144/235/290, 82/127/144/266, 82/127/144/266/290, 82/127/144/378, 82/127/235, 82/127/290, 82/144/266/290/378, 82/144/266/378, 82/235/266, 82/235/266/378/383, 82/235/290, 82/235/378, 82/266, 82/266/378, 82/378, 113/270/370/384, 115/256/333/375, 115/256/375, 115/273/375, 115/375, 127/144/290, 127/149/150, 127/266/290/378, 127/378/383, 144/235/290, 144/266/290, 233/334, 233/376, 235, 235/290, 256/333, 262, 266/378, 270/370, 273, 276/352, 290, 290/378, 333, 358, 370, 375, and 378 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2540.
32. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 18, 18/113, 18/113/252, 18/113/252/336/384, 18/113/252/370, 18/113/270, 18/113/270/370/384, 18/259/370/384, 18/270, 18/270/370, 18/270/370/384, 18/370, 18/370/384, 73, 73/115/184/341 , 73/115/341 , 73/131/256/273/333/341/375, 73/184/256, 73/184/256/341 , 73/184/256/375, 73/184/273/333, 73/184/333, 73/184/341/375, 73/256, 73/256/273, 73/256/273/375, 73/256/333/375, 73/256/375, 73/273, 73/273/333/341 , 73/273/333/375, 73/273/375, 73/333, 73/333/341/375, 73/333/375, 73/341 , 73/375, 73/379, 77, 77/118, 77/118/143/262/276/334/376, 77/118/143/334/352/376, 77/118/143/376, 77/118/233, 77/118/233/352/376, 77/118/262/334/376, 77/118/334, 77/143/262/276, 77/143/262/334, 77/143/376, 77/233/238/376, 77/233/262, 77/233/276/376, 77/233/334/376, 77/262/376, 77/276, 77/334/376, 77/352, 82, 82/127, 82/127/144/235/290, 82/127/144/266, 82/127/144/266/290, 82/127/144/378, 82/127/235, 82/127/235/266/358, 82/127/290, 82/127/290/358, 82/127/290/378, 82/144/266/290/378, 82/144/266/378, 82/150/235/266, 82/235/266, 82/235/266/290, 82/235/266/378/383, 82/235/290, 82/235/378, 82/266, 82/266/378, 82/290, 82/378, 113, 113/252, 113/252/270/370, 113/270/370/384, 113/336, 118/233/276/334, 118/334, 127/235/266, 127/266/290/378, 127/378/383, 143, 143/276, 143/276/334, 144/235/290, 144/266/290, 144/290/358, 233/334, 233/376, 235, 235/266/290/358/383, 235/358, 252/329/336/370, 252/384, 256/333, 256/375, 262, 262/276, 262/334, 266/290, 266/378, 270/370, 273, 276/352, 290, 290/378, 333, 334, 334/376, 336/370, 341 , 358, 370, 375, and 378 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2540.
33. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 18,
18/77/118/169/233/238/256/266/270, 18/77/150/256/376, 18/118, 18/118/143/169/238/256/266/333, 18/118/150/169/184/238/256/270/378, 18/118/169, 18/118/233/238, 18/143, 18/143/256, 18/150/169, 18/150/266, 18/169/184/290/333/376, 18/169/233/333, 18/169/392, 18/184/270, 18/233/256/334, 18/256/266/290/378, 77/113/118/256/266/290, 77/118/290, 77/169/270/290, 77/233, 77/233/256, 77/238, 113/118/169/184/256/376, 113/169, 113/233, 118, 118/143/238, 118/233/238/256/378, 118/233/238/270, 143/169/233/270, 143/233/266/290/376, 143/238/270/290/378, 143/256/334, 143/290, 169, 169/256/266, 183/270/333, 233/238, 233/238/376, 233/392, 238/256, 256/290/303, 270, 290, 376, and 378 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3402.
34. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 18, 18/77/118/169/266/334/376, 18/77/150/256/376, 18/77/256/290/334, 18/113/238, 18/118, 18/118/143/169/238/256/266/333,
18/118/143/169/376, 18/118/169, 18/118/233/238, 18/118/266, 18/143, 18/143/169/233/238/358, 18/143/233/238/290/376, 18/143/256, 18/143/290, 18/143/333/334/378, 18/150/169, 18/150/266, 18/169/184/290/333/376, 18/169/233/333, 18/169/238/270/334/376, 18/169/392, 18/233/256/334, 18/233/266/290/376, 18/256/266/290/378, 18/266/270/378, 18/378, 77/113/121/143/233/334, 77/118/169/233/266/270, 77/118/238/376, 77/118/290, 77/169/270/290, 77/233, 77/238, 113/238/376, 118, 118/143/238, 118/143/392, 118/169/184/233/266/270/333, 118/233/238/256/378, 118/256/290/333/334/376/378, 118/256/334/376, 118/266/290, 143/169/233/270, 143/233/266/290/376, 143/238/270/290/378, 143/256/334, 143/290, 143/364, 150/233/333, 169, 169/256/266, 183/270/333, 233/238, 233/238/376, 233/392, 238/256, 238/266/270/378, 256/266/334, 266/270/376, 266/333, and 378 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3402.
35. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 77/156, 77/156/270, 77/184/244, 77/184/333, 77/244/270, 77/270, 143, 143/169/238/257/344, 143/169/255, 143/169/255/257, 143/169/343, 143/255, 143/255/257, 143/255/344, 143/257, 156, 156/270, 169/238, 169/238/255/257/344, 169/257/344, 169/344, 184, 184/244/324, 238, 238/255/344, 244/270, 257/344, and 270 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3662.
36. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 77, 77/156/184/244/324, 77/184/270, 143, 143/238, 143/238/255, 143/238/255/343, 143/238/255/392, 143/238/344, 143/255, 143/257/392,
143/344 , 169, 169/238/255/344, 169/238/344, 169/255, 169/343, 169/392, 238/344, 255, 255/257, 255/257/392, 257, and 344 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3662.
37. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 20, 22, 26, 42, 43, 44, 45, 50, 51 , 52, 53, 54, 55, 60, 62, 64, 65, 95, 97, 99, 103, 104, 111 , 112, 115, 115/143/169, 115/169/343, 115/343, 116, 116/343, 143, 143/169, 154/439, 169, 169/324, 169/324/343, 169/343, 186, 234, 236, 238, 238/246, 239, 239/244, 241 , 242, 243, 244, 250, 251 , 254, 255, 260, 283, 284, 295, 296, 298/326, 319, 322, 323, 324, 324/343, 326, 328, 329, 333, 334, 338, 340, 341 , 343, 344, 347, 366, 434, 439, 442, 443, 444, 445, 446, and 447 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3788.
38. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 26, 26/236, 26/236/260/333, 26/236/295, 26/260, 26/260/295, 26/260/295/333, 26/295/319, 26/333, 64, 64/244, 64/251/260/347, 64/260/295/333, 64/260/347, 103/115/255, 103/115/343, 103/115/343/344, 103/143/255/284/343/344, 103/169/343/344, 103/255/284, 103/343/344, 115, 115/143/255, 115/143/255/343, 115/143/284,
115/143/343, 115/143/344, 115/169, 115/169/343, 115/255, 115/255/344, 115/284, 115/284/343/344, 115/284/344, 115/343, 115/343/344, 115/344, 143, 143/169/255/284/343/344, 143/169/343/344, 143/255, 143/255/284/343/344, 143/255/343, 143/255/343/344, 143/255/344, 143/343/344, 169, 169/255, 169/255/284, 169/255/343, 169/284, 169/343, 169/344, 236, 236/244/333, 236/260, 236/347, 244, 244/251 , 244/251/319, 244/260, 244/295, 244/347, 251 , 251/260/295/347, 251/333, 255, 255/284, 255/343, 255/343/344, 255/344, 259, 260, 260/295, 260/333, 284, 284/343/344, 284/344, 295, 295/333, 333, 343, 343/344, 344, and 347 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4150.
39. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 115, 115/117, 115/117/118,
115/117/118/152, 115/117/118/169, 115/117/118/246, 115/117/152, 115/117/152/169, 115/117/152/169/246, 115/117/152/246, 115/117/169, 115/117/246, 115/118, 115/118/152, 115/118/152/169, 115/118/152/169/246, 115/118/169, 115/118/246, 115/152, 115/152/169, 115/152/169/246, 115/152/246, 115/152/246/374, 115/169, 115/169/246, 115/246, 117, 117/118, 117/118/152, 117/118/152/169, 117/118/152/246, 117/118/169, 117/118/246, 117/152, 117/152/169, 117/152/246/288, 117/169, 117/246, 118/246, 152, 152/169, 152/169/246, 152/246, 169, 169/246, and 246 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4278.
40. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 73, 101 , 114, 115, 118, 143, 145, 150, 151 , 152, 153, 154, 156, 157, 160, 161 , 163, 166, 167, 168, 169, 171 , 172, 190, 191 , 192, 195, 213, 214, 218, 219, 223, 225, 233, 233/343, 234, 235, 236, 237, 238, 239, 239/250, 243, 244, 249, 251 , 254, 255, 257, 258, 259, 261 , 262, 262/283, 277/292, 283, 284, 286, 289, 292, 295, and 296 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4278.
41 . The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 93, 113, 113/114, 113/115, 113/143, 115/118, 115/177, 118/143/163/168/195/255/315, 118/143/255, 118/163/168/214/255/315, 118/168/214/315, 118/255, 124, 126, 129, 134, 143/154/255, 143/156/168/216/255, 143/163, 143/163/255, 143/168, 143/168/169/255/315, 143/255, 148, 156/214/315, 159, 163, 163/168, 163/168/169, 163/168/169/255/315, 163/168/169/315, 163/168/195/214/255/315, 163/214, 163/255, 163/255/315, 168/315, 186, 194, 197, 206, 207, 208, 210, 213, 255/315, 277, 293, 294, 315, 317, 320, 322, 328, 329, 330, 333, 352, 366, 392, and 445 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4642.
42. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 127, 128, 129, 140, 180, 182, 194, 277, 293, 294, 299, 300, 317, 319, 320, 324, 333, 356, 366, and 392 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4642.
43. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 1 13, 1 17, 1 18, 151 , 191 , 196, and 205 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4984.
44. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 1 14/1 15/143, 1 14/1 15/143/233, 1 14/1 15/233, 1 14/1 15/343, 1 14/143, 1 14/143/151/233/238/261 , 1 14/143/213/261/343, 1 14/143/233, 1 14/143/233/238, 1 14/151/261/343, 1 14/213/238, 1 14/343, 1 15, 1 15/143, 1 15/143/151/154/213/261 ,
1 15/143/151/154/233/343, 1 15/143/151/233/261 , 1 15/143/151/233/343, 1 15/143/154/213/233/238/261 , 1 15/143/213, 1 15/143/233, 1 15/143/343, 1 15/151/154/233, 1 15/151/261 , 1 15/213/233, 1 15/213/233/238, 1 15/213/261 , 1 15/233/238, 1 15/238, 1 15/343, 1 15/348, 143, 143/151/154, 143/151 /154/213, 143/154, 143/213/233, 143/213/233/238/261/343, 143/213/261/343, 143/233/261 , 143/261 , 151/154, 151/154/213/343, 151/154/343, 151 /213, 151/213/233, 213/233/343, and 233/238/343 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4984.
45. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 1 17, 1 17/1 18/143/168/169/392, 1 17/1 18/143/392, 1 17/1 18/168/169, 1 17/1 18/168/169/392, 1 17/143/168/169/170/392,
1 17/143/168/169/392, 1 17/168/169, 1 17/168/169/392, 1 17/168/392, 1 17/169, 1 17/392, 1 18/143, 1 18/143/168/169, 1 18/143/169, 1 18/143/392, 1 18/168, 1 18/168/392, 1 18/169/392, 1 18/392, 143/168/169/392, 143/169, 143/392, 168/169, 168/169/392, 168/392, 169/392, and 392 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5196.
46. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 134, 134/182/194/294, 134/182/277/293, 134/182/293/320, 134/182/294, 134/194/277, 134/194/277/317, 134/194/293, 134/194/293/294, 134/194/293/317, 134/194/294, 134/194/317, 134/194/317/320, 134/317, 182, 182/194, 182/194/293/320, 182/293/294, 194, 194/277/293/317, 194/277/317/320, 194/277/320, 194/293/294/317, 194/293/320, 277, 293, 317, 317/320, and 320 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5286.
47. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 12/134, 13/134, 13/134/210, 22/134/152, 22/134/235/344, 64/93/134/235, 64/134/344/347, 77/134, 79/134/210, 84/134, 86/134, 87/134, 89/134, 93/134/152/235/344, 93/134/235/344, 93/134/244/344, 93/134/344, 131 , 134/152/344, 134/175, 134/210/382, 134/210/402, 134/210/456, 134/210/478, 134/344, 134/377, 134/378, 134/380, 134/381 , 134/382, 134/384, 134/386, 134/387, 134/388, 134/390, 134/391 , 134/393, 134/395, 134/398, 134/401 , 134/402, 134/406, 134/407, 134/421 , 134/456, 134/457, 134/459, 134/460, 134/464, 134/467, 134/468, 134/470, 134/471 , 134/473, 134/473/474, 134/474, and 134/475 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5412.
48. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 73/127/134/160/172/198/290,
73/127/134/160/198/333, 73/127/134/160/210/266, 73/127/134/172/192/266/333, 73/127/134/172/198, 73/127/134/210/290, 73/127/134/210/333, 73/127/134/290/333, 73/127/134/333, 73/134, 73/134/160/172/192/290, 73/134/160/172/198/333, 73/134/160/172/290, 73/134/160/266, 73/134/160/333, 73/134/172, 73/134/172/210/333, 73/134/172/266/333, 73/134/198/210, 73/134/198/210/266, 73/134/210/333, 73/134/333, 73/134/366, 127/134, 127/134/160/172/192/333, 127/134/160/172/210/266/290, 127/134/172/198/266/333, 127/134/192/210, 127/134/198/210/290, 127/134/210/266/290, 127/134/210/333, 134, 134/160/172/333, 134/160/198/266/290, 134/172/192/290/333, 134/192/198, 134/198, 134/198/266/333, 134/210, 134/210/290, and 134/333 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5412.
49. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 13, 13/178/381 , 13/178/381/473, 13/178/457, 13/381 , 13/381 /457, 13/402/457, 13/457, 178, 178/381/457, 178/381/457/473, 178/402/457, 178/473, 381 , 381/393/457, 381/457/473, 381/473, and 393/457 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5422.
50. The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from
38/73/1 13/1 15/1 17/1 18/127/134/152/154/160/163/164/168/169/172/184/192/194/198/220/243/260/266/27 0/295/317/333/392/423, 38/73/1 13/1 15/1 17/1 18/127/134/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/29 5/317/333/392/423,
38/73/1 13/1 15/1 17/1 18/127/134/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/29 5/317/333/392/423/457,
38/73/1 13/1 15/1 17/1 18/127/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/295/31 7/333/392/423,
38/73/1 13/1 15/1 18/127/152/154/160/162/164/168/172/184/192/198/220/243/260/266/270/295/317/333/42 3,
38/73/1 13/1 15/1 18/127/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/295/317/33 3/423,
38/73/1 13/1 15/1 18/127/152/154/160/164/168/172/184/189/192/198/220/243/260/266/270/295/317/333/42 3,
38/73/1 13/1 15/1 18/127/152/154/160/164/168/172/184/192/198/210/220/243/260/266/270/295/317/333/42 3, 38/73/1 13/1 15/1 18/127/152/154/160/164/168/172/184/192/198/220/243/260/266/270/295/317/333/423, 38/73/1 13/1 18/127/152/154/160/164/168/172/184/192/198/220/243/260/266/270/295/317/333/423, 38/73/1 13/1 18/127/152/154/160/164/168/172/184/192/198/220/243/266/270/317/333/423, 38/73/1 13/1 18/127/152/154/160/164/168/172/184/192/198/220/266/270/317/333/423, and
38/1 13/127/152/154/160/164/168/172/198/220/290/317/423, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972.
51 . The engineered transposase of Claim 1 , wherein at least one substitution or substitution set comprises substitutions at amino acid positions selected from 48, 1 16, 1 18, 120, 130, 137, 153, 157,
163, 175, 181 , 187, 192, 213, 246, 263, 273, 296, 318, 334, 341 , 394, 412, 424, 454, and 458, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5704.
52. The engineered transposase of any of Claims 1 -51 , wherein said engineered transposase is capable of binding to adapters or donor DNA to form a transposome, cleaving or fragmenting target DNA, and/or ligating the adapters or donor DNA to the target DNA.
53. The engineered transposase of any of Claims 1 -52, having at least one improved property, as compared to a wild-type or reference transposase.
54. The engineered transposase of Claim 53, wherein said improved property is selected from increased polynucleotide fragmentation or cleavage activity, increased thermostability, increased activity at elevated temperatures, increased soluble expression, decreased inhibition, increased adapter loading or adapter ligation (i.e. tagging), increased binding to a target polynucleotide, and/or decreased sequence insertion bias.
55. The engineered transposase of any of Claims 1 -54, wherein said engineered transposase comprises increased polynucleotide fragmentation or cleavage activity as compared to a wild-type or reference transposase.
56. The engineered transposase of any of Claims 1 -54, wherein said engineered transposase comprises increased thermal stability as compared to a wild-type or reference transposase.
57. The engineered transposase of any of Claims 1 -54, wherein said engineered transposase comprises increased activity at elevated temperatures as compared to a wild-type or reference transposase.
58. The engineered transposase of any of Claims 1 -54, wherein said engineered transposase comprises increased adapter loading or adapter ligation (i.e. tagging) as compared to a wild-type or reference transposase.
59. The engineered transposase of any of Claims 1 -54, wherein said engineered transposase comprises increased binding to a target polynucleotide, as compared to a wild-type or reference transposase.
60. The engineered transposase of any of Claims 1 -54, wherein said engineered transposase comprises decreased sequence insertion bias, as compared to a wild-type or reference transposase.
61 . The engineered transposase of any of Claims 1 -60, wherein said engineered transposase comprises a polypeptide sequence selected from the polypeptide sequences between SEQ ID NOs: 2- 2368, 2387-5694, and 5706-5756.
62. The engineered transposase of any of Claims 1 -61 , wherein said transposase is purified.
63. A composition comprising at least one engineered transposase of any of Claims 1 -62.
64. A polynucleotide sequence encoding at least one engineered transposase of any of Claims 1 -61 .
65. A polynucleotide sequence comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NOs: 1 , 3, 155, 301 , 337, 605, 971 , 1081 , 1217, 1579, 1597, 1599, 1603, 1719, 2367, 2425, 2539, 3401 , 3661 , 3787, 4149, 4277, 4641 , 4983, 5195, 5285, 5411 , 5421 , and/or 5703, and/or or a functional fragment thereof, wherein said polynucleotide sequence encodes an engineered polypeptide comprising at least one substitution at one or more amino acid positions.
66. The polynucleotide sequence of Claim 65, wherein said polynucleotide sequence encodes at least one engineered transposase comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704.
67. The polynucleotide sequence of Claim 65, wherein said sequence comprises SEQ ID NOs: 1 , 3, 155, 301 , 337, 605, 971 , 1081 , 1217, 1579, 1597, 1599, 1603, 1719, 2367, 2425, 2539, 3401 , 3661 , 3787, 4149, 4277, 4641 , 4983, 5195, 5285, 5411 , 5421 , and/or 5703.
68. The polynucleotide sequence of Claim 64, wherein said polynucleotide sequence is operably linked to a control sequence.
69. The polynucleotide sequence of Claim 64, wherein said polynucleotide sequence is codon-optimized.
70. An expression vector comprising at least one polynucleotide sequence of any of Claims 64-69.
71 . A host cell comprising at least one expression vector of Claim 70.
72. A method of producing an engineered transposase polypeptide in a host cell comprising culturing a host cell of Claim 71 , under suitable culture conditions, such that at least one engineered transposase is produced.
73. The method of Claim 72, further comprising recovering at least one engineered transposase from the culture and/or host cells.
74. The method of Claim 72 or 73, further comprising the step of purifying said at least one engineered transposase.
75. A method for generating a mixture of altered nucleic acids comprising exposing a target nucleic acid to an engineered transposase under conditions and for a time sufficient for the engineered transposase to carry out a transposition event, wherein the engineered transposase comprises a polypeptide sequence comprising at least 85% sequence identity to a reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, or a functional fragment thereof, wherein the engineered transposase comprises at least one substitution or substitution set in its polypeptide sequence relative to the reference sequence.
76. The method of Claim 75, wherein the altered nucleic acids are fragmented from the target nucleic acid.
77. A method of preparing a nucleic acid library comprising:
(a) contacting a target nucleic acid with an engineered transposase under conditions and for a time sufficient for the engineered transposase to carry out a transposition event, wherein the engineered transposase comprises a polypeptide sequence comprising at least 85% sequence identity to a reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, or a functional fragment thereof, wherein the engineered transposase comprises at least one substitution or substitution set in its polypeptide sequence relative to the reference sequences;
(b) fragmenting the target nucleic acid into a plurality of nucleic acid fragments;
(c) selecting a subset of nucleic acid fragments from the plurality of nucleic acid fragments; and
(d) amplifying the subset of nucleic acid fragments to generate the nucleic acid library.
78. A method of sequencing a target nucleic acid comprising:
(a) contacting the target nucleic acid with an engineered transposase under conditions and for a time sufficient for the engineered transposase to carry out a transposition event, wherein the engineered transposase comprises a polypeptide sequence comprising at least 85% sequence identity to a reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, and/or 5704, or a functional fragment thereof, wherein the engineered transposase comprises at least one substitution or substitution set in its polypeptide sequence relative to the reference sequence;
(b) fragmenting the target nucleic acid into a plurality of nucleic acid fragments;
(c) selecting a subset of nucleic acid fragments from the plurality of nucleic acid fragments;
(d) amplifying the subset of nucleic acid fragments to generate a nucleic acid library; and
(e) sequencing the nucleic acid library.
79. The method of Claim 77 or 78, further comprising adding a polynucleotide to the 5’ and/or 3’ end of each nucleic acid fragment of the plurality of nucleic acid fragments.
80. The method of any one of Claims 75-78, wherein the engineered transposase comprises a polypeptide sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NOs: 2, 4, 156, 302, 338, 606, 972, 1082, 1218, 1580, 1598, 1600, 1604, 1720, 2368, 2426, 2540, 3402, 3662, 3788, 4150, 4278, 4642, 4984, 5196, 5286, 5412, 5422, or 5704.
81 . The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 2 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 10, 13, 24, 35, 39, 44, 46, 51 , 54, 55, 62/361 , 72, 75, 82, 89, 92, 107, 128, 137, 164, 167, 177/359, 191 , 192, 193, 194, 201 , 217, 281 , 289, 291 , 291/340, 305, 309, 310, 317, 331 , 337, 340, 346, 352, 353, 357, 358, 359, 362, 371 , 396, 414/435, 415, 428, 437, 443, 447, and 448, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2.
82. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 4 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 4, 5, 6, 8, 10, 10/317, 10/317/358, 10/317/359, 10/346, 10/346/359, 10/358, 10/359, 12,
12/373, 13, 15, 16, 18, 19, 26, 30, 30/154, 31 , 34, 35, 37, 38, 68, 70, 73, 74, 93, 106, 118, 126, 134, 135, 143, 146, 154/453, 163, 182, 207/286, 208, 226, 237, 246, 247, 259, 262, 263, 264, 280, 282, 286, 287, 288, 289, 291 , 292, 293, 298, 305, 309, 310, 313, 317, 317/346, 317/359, 317/445, 320, 346, 351 , 355, 356, 358, 359, 360, 368, 369, 410, 411 , 422, 426, 427, 428, 446, 448, 453, and 454, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4.
83. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 156 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 18/73/74/259, 18/73/286/289/411 , 18/74/118/259/286/289/411 , 18/74/259,
18/74/259/286/289, 18/74/259/286/411 , 18/118/259, 18/259/286/289/411 , 18/259/289/411 , 18/289/411 , 73/74/259/286/289, 73/74/259/289, 73/259/286, 74/259, 74/259/289, 74/286/289/411 , 259, 259/286/289, and 289, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 156.
84. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 302 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 16/19/355/427, 16/37/146/286/287/289/310/313/355, 16/146/226/287/289/355, 19/37/146/286/287/310/355, 19/135/286/287/289/355, 19/182/355, 37/70/226/310/313/355, 37/74/310/355/427, 37/135/146/182/226/286/287/355/368, 37/146/226/289/427, 37/146/287/289/427, 38, 41 , 42, 43, 44, 45, 47, 48, 50, 53, 54, 55, 70/74/226/286/287/289/368/427, 70/74/355/368, 70/135/146/150/182/286/289/313/427, 74/310/313/355, 74/310/355, 74/355/427, 101 , 103, 117, 135/226/286/289/355/368, 135/355, 146, 146/150/287/289/310/355, 146/182/355, 146/226/286/355, 146/286/287/289/355/427, 146/289, 146/427, 150/226/310/313, 182/226/355, 182/355, 244, 247, 249, 251 , 287/355/427, 300, 317/411 , 355, 393, 397, 398, 399, 401 , 405, 407, 408, 409, 411 , 413, 426, 429, 431 , 439, 444, 446, 450, 453, 454, 457, 459, 460, 464, 466, 467, 469, 470, 471 , 472, 474, 475, 476, and 478, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 302.
85. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 338 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 16, 16/313, 16/409/411 /427, 37/150/408, 37/313/317/408/411 , 37/317/405/408, 37/317/405/426, 37/405/409/427, 150, 150/313/405/411 , 150/405/406, 150/408, 150/408/411 , 226/405, 317/460, and 408/426, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 338.
86. The method of any one of claims 75-80, wherein the reference sequence is SEQ ID NO.
606 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 12, 13, 18, 19, 22, 26, 31 /262, 34, 60, 79, 80, 81 , 82, 92, 106, 107, 113, 115, 132, 134, 136,
140, 143, 152, 160, 163, 167, 168, 175, 179, 183, 195, 199, 201 , 202, 215, 216, 218, 220, 223, 226, 233,
235, 236, 237, 243, 255, 259, 261 , 262, 266, 268, 270, 273, 282, 290, 292, 293, 309, 319, 320, 326, 343,
346, 365, 370, 374, 380, 382, 386, 389, 390, 408, 412, 414, 415, 424, and 442, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 606.
87. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO.
606 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 2, 12, 13, 15, 16, 18, 19, 22, 26, 30, 31/262, 34, 51 , 60, 69, 79, 82, 84, 87, 88, 106, 107, 113, 114, 115, 128, 132, 134, 135, 136, 140, 143, 146, 150, 152, 160, 163, 167, 168, 175, 179, 183, 195,
198, 199, 201 , 215, 217, 218, 219, 220, 223, 224, 226, 227, 231 , 233, 235, 236, 243, 254, 255, 259, 261 ,
266, 267, 268, 270, 272, 273, 275, 277, 279, 282, 287, 289, 290, 291 , 292, 293, 304, 307, 309, 319, 320,
343, 344, 346, 355, 365, 370, 373, 380, 382, 386, 389, 390, 408, 412, 414, 415, 418, 423, 424, and 468, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 606.
88. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 606 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 44/45/47/408/453, 44/45/47/453, 44/45/47/453/454, 44/45/103/453/457, 44/45/453/454/457, 44/47/103/393/408/453/454, 44/47/103/453/454/457, 44/47/393/408/453/457, 44/47/393/453/457,
44/103/408/453/454, 45/47/103/393/453/454/457, 45/47/393/453/454, 45/47/408/423/453/457, 45/47/453, 45/103/393/453/454, 45/453/454, 47/103/408/453/454, 47/393/408/453/454/457, 47/393/453/454, 47/408/453/457, 53/54/244/450, 103/393/408/453/457, 244/450, 408/453/454, 446/450/464, and 453/457, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 606.
89. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 972 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 38/45/53, 38/45/53/54/476, 38/45/53/244, 38/45/53/470/472, 38/45/54/244, 38/45/54/470/474/478, 38/45/472/476/478, 38/45/476, 38/53/54, 38/53/54/244, 38/53/54/244/472/478, 38/53/472, 38/54/244/470/474/476/478, 38/244/470/472/474/478, 38/478, 45/53/54/244/470/474/476/478, 45/53/54/470/474/476, 45/53/54/472/478, 45/54/244/474/478, 53, 53/54/470/476/478, 53/244/474/478, 54, 54/244, 54/244/472/476, 54/472, 244, and 474/478, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972.
90. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 972 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 22/38/45/107/259/317/412, 22/38/45/259/317/412, 22/107, 22/107/317/412, 22/134, 22/134/220, 22/134/220/317, 22/134/317/412, 22/317, 38, 38/45/81/143/244, 38/53/243, 38/81 , 38/81/244, 38/107/134/220, 38/132, 38/220/317, 45/81 , 45/81/243, 45/81/243/343, 45/143/243/270/343, 45/244/270, 53/115/163/243/270/343, 53/143/243/270/343, 53/270, 64/134/220/317/412, 81/132/243, 107, 107/134/317, 115/244/270, 134/259, 143/243/270, 243, 243/270, 244, 244/270, 259, 270, and 317, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972.
91 . The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 972 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 22/38/45/107/259/317/412, 22/38/45/259/317/412, 22/38/64/134/220, 22/45/220/259, 22/64/107/259/317/412, 22/64/134/220, 22/107, 22/134, 22/134/317, 22/134/317/412, 22/220, 22/220/412/415, 22/259/317, 22/259/317/412, 22/317, 38, 38/45/81/143/244, 38/45/143/270, 38/53/243, 38/81 , 38/81/143/243/270, 38/81/183/243/270, 38/81/243, 38/81/244, 38/107/134/220, 38/132, 38/220/317, 38/270/343, 45/81 , 45/81/132/243/270, 45/81/243, 45/143/243/270/343, 45/244/270, 53/143/243/270/343, 53/244/270, 53/270, 64, 64/107/134, 64/107/317, 64/134, 64/134/220/317/412, 81/132, 81/132/243, 107, 107/134/317, 107/412, 115/244/270, 143/243/270, 220/259, 243, 243/270, 244/270, 255, 259, and 270, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972.
92. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 1082 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 143, 143/175/272/273/415, 152/168/216/237, 152/168/423, 160, 160/201/272, 160/201/273/415, 160/226, 160/244/272/273/415, 160/415, 168, 168/187/216/255/423, 168/216/237, 168/255/423, 168/290/423, 187, 187/423, 199/201/226, 199/201/415, 201 , 216, 216/423, 226/244/415, 226/415, 237, 237/290, 244, 255, 272, 290, 290/423, 415, and 423, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1082.
93. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 1082 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 143, 143/160/226/415, 143/175/272/273/415, 152/168/216/237, 152/168/423, 160,
160/201/272, 160/201/273/415, 160/226, 160/244/272/273/415, 160/415, 168, 168/187/216/255/423, 168/216/237, 168/216/237/255/343, 168/255/423, 168/290/423, 187, 199/201/226, 199/201/415, 201 , 216, 216/423, 226/244/415, 226/415, 237, 237/290, 244, 255, 255/343, 272, 290, 290/423, 415, and 423, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1082.
94. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 1082 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 72, 90, 91 , 92, 93, 95, 122, 129, 141 , 142, 143, 144, 145, 150, 151 , 151/155, 152, 154, 155, 157, 160, 161 , 161/313, 164, 165, 168, 180, 181 , 184, 193, 197, 204, 205, 205/472, 206, 299, and 320, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1082.
95. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 1082 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 90, 92, 122, 129, 141 , 143, 144, 145, 150, 151 , 151/155, 152, 154, 155, 155/265, 157, 160, 161 , 164, 165, 168, 180, 181 , 184, 193, 197, 204, 205, 205/472, 206, 299, and 320, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1082.
96. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 1218 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 129, 129/160, 129/160/164, 129/160/184, 129/164, 129/164/184, 129/205, 146/152/244, 150/155/226, 150/155/244, 152, 154, 154/160/184, 154/164, 154/205, 155, 160, 160/164, 160/164/184, 160/184, 164, 164/204, 164/205, 204, 205, 226, 226/244, and 244, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1218.
97. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 1218 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 129, 129/143, 129/154/164, 129/154/164/205, 129/154/205, 129/160, 129/160/164, 129/160/205, 129/164/205, 143, 143/150/155/226/244, 143/150/155/244, 143/150/226, 143/150/226/244/343, 143/150/226/343, 143/150/244, 143/150/343, 143/152, 143/152/226, 143/152/226/244, 143/152/226/343, 143/152/343, 143/155, 143/155/226/244, 143/155/226/244/343, 143/155/343, 143/226, 143/226/244/343, 143/226/343, 143/244, 143/244/343, 143/343, 146/152/244, 150/155/226, 150/155/244, 150/155/244/343, 150/226/244/343, 152, 152/174/244/343, 152/226, 152/226/343, 152/343, 154, 154/160/184, 154/164, 154/164/204, 154/164/204/205, 154/205, 155, 160,
160/164, 160/164/184, 164, 205, 226, 226/244, and 244, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1218.
98. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 1580 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 98/129/157/168, 129/157/168, 151 , 151/152/255, 151/152/273, 151/255, 151/343, 157, 157/164, 157/164/168, 157/168, 164, 164/255, 168, 183, 255, 255/343, and 343, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
99. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 1580 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 129, 129/157, 129/157/168, 129/164, 129/164/168/183, 129/164/183, 143, 143/151 , 143/151/152, 143/151/164, 143/164, 143/164/255, 143/164/343, 143/255, 143/343, 151 , 151/152, 151/152/164/343, 151/152/343, 151/255, 151/255/343, 151/343, 152, 152/255, 157, 157/164/168, 157/168, 164, 164/168, 164/255, 164/447, 168, 183, 255, 255/343, 298/343, and 343, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
100. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 1580 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 69, 69/164, 72, 73, 91 , 92/164, 95, 96, 97, 98, 110, 112, 113, 121 , 121/164, 122, 122/164, 124, 126, 127, 139, 140, 141 , 144, 144/164, 145, 146, 147, 148, 150, 150/164, 151 , 151/164, 153, 153/164, 155, 156, 157, 159, 160, 161 , 163, 164, 164/172, 164/173, 164/182, 164/188, 164/191 , 164/192, 164/195, 164/197, 164/199, 164/202, 164/205/249, 164/209, 165, 166, 167, 169, 170, 171 , 172, 173, 177, 184, 187, 188, 190, 191 , 192, 193, 194, 195, 197, 197/266, 198, 199, 200, 202, 203, 204, 205, 208, 209, 210, 321 , 324, 331 , and 349, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
101 . The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 1580 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 69, 69/164, 72, 73, 91 , 92/164, 95, 97, 98, 121/164, 126, 127, 139, 140, 142, 144, 144/164, 145, 146, 146/164, 148, 150, 150/164, 151 , 153, 153/164, 155, 156, 157, 160, 163, 164/173, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/205/249, 164/209, 165, 166, 167, 169, 171 , 172, 173, 177, 182, 187, 188, 190, 191 , 192, 194, 195, 197/266, 198, 199, 200, 202, 203, 204, 205, 208, 209, 210, 324, 331 , and 349, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
102. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 1580 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 73, 91 , 93, 95, 97, 98, 110, 112, 122, 122/164, 124, 127, 139, 144, 145, 146, 146/164, 147, 148, 150, 150/164, 151 , 151/164, 153, 153/164, 155, 156, 157, 159, 160, 161 , 162, 163, 164, 164/182, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/205/249, 164/209, 165, 166, 167, 169, 171 , 172, 177, 184, 187, 188, 190, 191 , 192, 193, 195, 197/266, 198, 199, 200, 204, 205, 206, 207, 208, 209, 210, and 324, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
103. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 1580 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 69, 73, 91 , 127, 140, 144, 145, 146, 146/164, 150, 150/164, 151 , 153, 155, 156, 157, 160, 163, 164, 164/172, 164/188, 164/191 , 164/192, 164/195, 164/199, 164/209, 165, 167, 169, 171 , 172, 182, 183, 188, 191 , 192, 193, 195, 196, 198, 199, 200, 204, 205, 208, 210, and 324, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 1580.
104. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 972 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 38/143/168/220/290/317/423, 38/152/154/164/168/220/290/317/423,
38/152/168/220/290/317/343/423, 38/152/168/220/290/317/423, 38/154/164/168/220/290/317/423, 38/168/220/290/317/423, and 38/220/317, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972.
105. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 2368 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 73/143/169/191/193, 73/156, 73/156/192/435, 73/156/210, 73/163, 73/163/210, 73/192/193, 127, 127/160/172/198, 127/172, 127/198, 146, 146/151/188, 146/324, 151/171/324, 151/324, 156/169, 156/192, 157/171 , 163, 163/169, 163/169/191/192/193, 165/171/324, 165/188/324, 165/324, 169, 171/188/324, 171/324, 172, 188, 192, 192/193, 198, 210, and 324 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2368.
106. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 2368 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 73, 73/163/191/192/193, 127/160/172, 127/160/172/198, 144, 160, 160/172, 163/191/192, and 192 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2368.
107. The method of any one of claims 75-80, wherein the reference sequence is SEQ ID NO. 2426 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 8, 13, 17, 18, 20/146/157/167, 26, 34/38, 38/39/45, 67, 70, 73, 73/150/155/169, 73/150/171/192, 73/155, 73/155/169/199, 73/155/171 , 73/192, 73/199, 78, 82, 84, 86, 86/384, 89, 90, 91 , 95, 102, 110, 111 , 113, 114, 115, 122/127, 126/127, 127/131/165, 127/133, 143, 143/146, 143/146/153, 143/146/157, 143/146/167, 143/146/167/188, 143/146/188, 143/153/167/188/246, 143/156/157/167/195, 143/156/188, 143/188, 143/188/195, 143/195, 144, 145, 145/151 , 146, 146/152, 146/153/157, 146/153/157/167, 146/153/167, 146/157, 146/157/167, 146/167, 150, 150/155/169, 150/155/169/192, 150/155/192, 150/169, 150/192, 150/199, 151/152/154, 152/154, 152/154/155/160, 153/157/167,
155/171 , 155/192, 156/270, 160/371 , 167/188, 168/172, 169, 184, 190, 192/198, 198, 206, 208, 209, 210, 214/220, 215/220, 216/220, 217/220, 219/220, 220, 220/222, 220/225, 220/226, 231 , 235, 242, 257, 259, 261 , 262, 267, 268, 269, 270, 272, 274, 275, 276, 277, 278, 282, 283/290, 284/290, 287/290, 288/290, 289/290, 290, 290/291 , 290/292, 290/293, 290/294, 304, 305, 305/373, 306, 307, 309, 317, 329, 333, 334, 339, 341 , 344, 345, 346, 352, 353, 355, 358, 366, 368, 370, 372, 373, 375, 376, 378, 379, 380, 381 , 382, 383, 384, 385, 386/393, 387/393, 389/393, 390/393, 391/393, 392/393, 393/394, 414, 415, 420/423, 422/423, 423, 423/425, 432, 440, 441 , 443, 469, and 472 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2426.
108. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 2426 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 8, 18, 25, 29, 30, 67, 73, 73/150/155/169, 73/150/155/171 , 73/150/171 /192, 73/171 , 73/192, 73/199, 74, 82, 86, 86/384, 113, 115, 118, 127/128, 143, 150, 150/155/169, 150/155/169/192, 150/155/171 , 150/155/171/199, 150/155/192, 150/155/199, 150/192, 150/199, 155/192, 168/171/172, 195/198, 197/198, 198/201 , 198/203, 198/205, 235, 252, 256, 257, 262, 263, 267/343, 268, 269, 270, 272, 273, 276, 277, 288/290, 289/290, 290, 290/291 , 290/292, 290/294, 298, 304, 305, 307, 333, 346, 355, 366, 370, 386/393, 389/393, 393/395, 440, and 472 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2426.
109. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 2540 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 66, 67, 70, 101 , 102, 103, 104, 106, 107, 108, 113, 114, 115, 117, 118, 143, 152, 154, 235, 236/251 , 237, 238, 242, 243, 246, 247, 249, 250, 251 , 256/352, 257, 259, 285, 286, 287, 288, 289, 290, 292, 293, 294, 295, 296, 322, 323, 326, 329, 330, 333, 339, 340, 343, 344, 345, and 352. and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2540.
110. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 2540 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 18, 18/113/270, 18/113/270/329/370, 18/113/270/370/384, 18/113/270/370/412, 18/113/329/384, 18/259/370/384, 18/270, 18/270/370, 18/270/370/384, 18/370, 18/370/384, 73, 73/115, 73/115/184/333, 73/115/184/341 , 73/115/256, 73/184/256, 73/184/273/333, 73/184/333, 73/256, 73/256/375, 73/273, 73/273/333/375, 73/273/375, 73/333, 73/333/375, 73/375, 73/379, 77/233/238/376, 77/233/334/376, 77/262/376, 77/276, 77/334/376, 82, 82/127, 82/127/144/235/290, 82/127/144/266, 82/127/144/266/290, 82/127/144/378, 82/127/235, 82/127/290, 82/144/266/290/378, 82/144/266/378, 82/235/266, 82/235/266/378/383, 82/235/290, 82/235/378, 82/266, 82/266/378, 82/378, 113/270/370/384, 115/256/333/375, 115/256/375, 115/273/375, 115/375, 127/144/290, 127/149/150, 127/266/290/378, 127/378/383, 144/235/290, 144/266/290, 233/334, 233/376, 235, 235/290, 256/333, 262, 266/378, 270/370, 273, 276/352, 290, 290/378, 333, 358, 370, 375, and 378 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2540.
111. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 2540 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 18, 18/113, 18/113/252, 18/113/252/336/384, 18/113/252/370, 18/113/270, 18/113/270/370/384, 18/259/370/384, 18/270, 18/270/370, 18/270/370/384, 18/370, 18/370/384, 73, 73/115/184/341 , 73/115/341 , 73/131/256/273/333/341 /375, 73/184/256, 73/184/256/341 , 73/184/256/375, 73/184/273/333, 73/184/333, 73/184/341/375, 73/256, 73/256/273, 73/256/273/375, 73/256/333/375, 73/256/375, 73/273, 73/273/333/341 , 73/273/333/375, 73/273/375, 73/333, 73/333/341/375, 73/333/375, 73/341 , 73/375, 73/379, 77, 77/118, 77/118/143/262/276/334/376, 77/118/143/334/352/376, 77/118/143/376, 77/118/233, 77/118/233/352/376, 77/118/262/334/376, 77/118/334, 77/143/262/276, 77/143/262/334, 77/143/376, 77/233/238/376, 77/233/262, 77/233/276/376, 77/233/334/376, 77/262/376, 77/276, 77/334/376, 77/352, 82, 82/127, 82/127/144/235/290, 82/127/144/266, 82/127/144/266/290, 82/127/144/378, 82/127/235, 82/127/235/266/358, 82/127/290, 82/127/290/358, 82/127/290/378, 82/144/266/290/378, 82/144/266/378, 82/150/235/266, 82/235/266, 82/235/266/290, 82/235/266/378/383, 82/235/290, 82/235/378, 82/266, 82/266/378, 82/290, 82/378, 113, 113/252, 113/252/270/370, 113/270/370/384, 113/336, 118/233/276/334, 118/334, 127/235/266, 127/266/290/378, 127/378/383, 143, 143/276, 143/276/334, 144/235/290, 144/266/290, 144/290/358, 233/334, 233/376, 235, 235/266/290/358/383, 235/358, 252/329/336/370, 252/384, 256/333, 256/375, 262, 262/276, 262/334, 266/290, 266/378, 270/370, 273, 276/352, 290, 290/378, 333, 334, 334/376, 336/370, 341 , 358, 370, 375, and 378 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2540.
112. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 3402 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 18, 18/77/118/169/233/238/256/266/270, 18/77/150/256/376, 18/118,
18/118/143/169/238/256/266/333, 18/118/150/169/184/238/256/270/378, 18/118/169, 18/118/233/238, 18/143, 18/143/256, 18/150/169, 18/150/266, 18/169/184/290/333/376, 18/169/233/333, 18/169/392, 18/184/270, 18/233/256/334, 18/256/266/290/378, 77/113/118/256/266/290, 77/118/290, 77/169/270/290, 77/233, 77/233/256, 77/238, 113/118/169/184/256/376, 113/169, 113/233, 118, 118/143/238, 118/233/238/256/378, 118/233/238/270, 143/169/233/270, 143/233/266/290/376, 143/238/270/290/378, 143/256/334, 143/290, 169, 169/256/266, 183/270/333, 233/238, 233/238/376, 233/392, 238/256, 256/290/303, 270, 290, 376, and 378 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3402.
113. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 3402 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 18, 18/77/118/169/266/334/376, 18/77/150/256/376, 18/77/256/290/334, 18/113/238,
18/118, 18/118/143/169/238/256/266/333, 18/118/143/169/376, 18/118/169, 18/118/233/238, 18/118/266, 18/143, 18/143/169/233/238/358, 18/143/233/238/290/376, 18/143/256, 18/143/290, 18/143/333/334/378, 18/150/169, 18/150/266, 18/169/184/290/333/376, 18/169/233/333, 18/169/238/270/334/376, 18/169/392, 18/233/256/334, 18/233/266/290/376, 18/256/266/290/378, 18/266/270/378, 18/378, 77/113/121/143/233/334, 77/118/169/233/266/270, 77/118/238/376, 77/118/290, 77/169/270/290, 77/233, 77/238, 113/238/376, 118, 118/143/238, 118/143/392, 118/169/184/233/266/270/333, 118/233/238/256/378, 118/256/290/333/334/376/378, 118/256/334/376, 118/266/290, 143/169/233/270, 143/233/266/290/376, 143/238/270/290/378, 143/256/334, 143/290, 143/364, 150/233/333, 169, 169/256/266, 183/270/333, 233/238, 233/238/376, 233/392, 238/256, 238/266/270/378, 256/266/334, 266/270/376, 266/333, and 378 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3402.
114. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 3662 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 77/156, 77/156/270, 77/184/244, 77/184/333, 77/244/270, 77/270, 143, 143/169/238/257/344, 143/169/255, 143/169/255/257, 143/169/343, 143/255, 143/255/257, 143/255/344, 143/257, 156, 156/270, 169/238, 169/238/255/257/344, 169/257/344, 169/344, 184, 184/244/324, 238, 238/255/344, 244/270, 257/344, and 270 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3662.
115. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 3662 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 77, 77/156/184/244/324, 77/184/270, 143, 143/238, 143/238/255, 143/238/255/343, 143/238/255/392, 143/238/344, 143/255, 143/257/392, 143/344, 169, 169/238/255/344, 169/238/344, 169/255, 169/343, 169/392, 238/344, 255, 255/257, 255/257/392, 257, and 344 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3662.
116. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 3788 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 20, 22, 26, 42, 43, 44, 45, 50, 51 , 52, 53, 54, 55, 60, 62, 64, 65, 95, 97, 99, 103, 104, 111 , 112, 115, 115/143/169, 115/169/343, 115/343, 116, 116/343, 143, 143/169, 154/439, 169, 169/324, 169/324/343, 169/343, 186, 234, 236, 238, 238/246, 239, 239/244, 241 , 242, 243, 244, 250, 251 , 254, 255, 260, 283, 284, 295, 296, 298/326, 319, 322, 323, 324, 324/343, 326, 328, 329, 333, 334, 338, 340, 341 , 343, 344, 347, 366, 434, 439, 442, 443, 444, 445, 446, and 447 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 3788.
117. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 4150 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 26, 26/236, 26/236/260/333, 26/236/295, 26/260, 26/260/295, 26/260/295/333, 26/295/319, 26/333, 64, 64/244, 64/251/260/347, 64/260/295/333, 64/260/347, 103/115/255, 103/115/343, 103/115/343/344, 103/143/255/284/343/344, 103/169/343/344, 103/255/284, 103/343/344, 115,
115/143/255, 115/143/255/343, 115/143/284, 115/143/343, 115/143/344, 115/169, 115/169/343, 115/255, 115/255/344, 115/284, 115/284/343/344, 115/284/344, 115/343, 115/343/344, 115/344, 143, 143/169/255/284/343/344, 143/169/343/344, 143/255, 143/255/284/343/344, 143/255/343, 143/255/343/344, 143/255/344, 143/343/344, 169, 169/255, 169/255/284, 169/255/343, 169/284, 169/343, 169/344, 236, 236/244/333, 236/260, 236/347, 244, 244/251 , 244/251/319, 244/260, 244/295, 244/347, 251 , 251/260/295/347, 251/333, 255, 255/284, 255/343, 255/343/344, 255/344, 259, 260, 260/295, 260/333, 284, 284/343/344, 284/344, 295, 295/333, 333, 343, 343/344, 344, and 347 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4150.
118. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO.
4278 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 115, 115/117, 115/117/118, 115/117/118/152, 115/117/118/169, 115/117/118/246, 115/117/152, 115/117/152/169, 115/117/152/169/246, 115/117/152/246, 115/117/169, 115/117/246, 115/118, 115/118/152, 115/118/152/169, 115/118/152/169/246, 115/118/169, 115/118/246, 115/152, 115/152/169, 115/152/169/246, 115/152/246, 115/152/246/374, 115/169, 115/169/246, 115/246, 117, 117/118, 117/118/152, 117/118/152/169, 117/118/152/246, 117/118/169, 117/118/246, 117/152, 117/152/169, 117/152/246/288, 117/169, 117/246, 118/246, 152, 152/169, 152/169/246, 152/246, 169, 169/246, and 246 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4278.
119. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 4278 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 73, 101 , 114, 115, 118, 143, 145, 150, 151 , 152, 153, 154, 156, 157, 160, 161 , 163, 166,
167, 168, 169, 171 , 172, 190, 191 , 192, 195, 213, 214, 218, 219, 223, 225, 233, 233/343, 234, 235, 236, 237, 238, 239, 239/250, 243, 244, 249, 251 , 254, 255, 257, 258, 259, 261 , 262, 262/283, 277/292, 283, 284, 286, 289, 292, 295, and 296 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4278.
120. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 4642 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 93, 113, 113/114, 113/115, 113/143, 115/118, 115/177, 118/143/163/168/195/255/315, 118/143/255, 118/163/168/214/255/315, 118/168/214/315, 118/255, 124, 126, 129, 134, 143/154/255, 143/156/168/216/255, 143/163, 143/163/255, 143/168, 143/168/169/255/315, 143/255, 148, 156/214/315, 159, 163, 163/168, 163/168/169, 163/168/169/255/315, 163/168/169/315, 163/168/195/214/255/315,
163/214, 163/255, 163/255/315, 168/315, 186, 194, 197, 206, 207, 208, 210, 213, 255/315, 277, 293, 294, 315, 317, 320, 322, 328, 329, 330, 333, 352, 366, 392, and 445 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4642.
121 . The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 4642 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 127, 128, 129, 140, 180, 182, 194, 277, 293, 294, 299, 300, 317, 319, 320, 324, 333, 356, 366, and 392 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4642.
122. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 4984 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 1 13, 1 17, 1 18, 151 , 191 , 196, and 205 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4984.
123. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 4984 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 1 14/1 15/143, 1 14/1 15/143/233, 1 14/1 15/233, 1 14/1 15/343, 1 14/143, 1 14/143/151/233/238/261 , 1 14/143/213/261/343, 1 14/143/233, 1 14/143/233/238, 1 14/151/261/343, 1 14/213/238, 1 14/343, 1 15, 1 15/143, 1 15/143/151/154/213/261 , 1 15/143/151/154/233/343,
1 15/143/151/233/261 , 1 15/143/151/233/343, 1 15/143/154/213/233/238/261 , 1 15/143/213, 1 15/143/233, 1 15/143/343, 1 15/151/154/233, 1 15/151/261 , 1 15/213/233, 1 15/213/233/238, 1 15/213/261 , 1 15/233/238, 1 15/238, 1 15/343, 1 15/348, 143, 143/151/154, 143/151/154/213, 143/154, 143/213/233, 143/213/233/238/261/343, 143/213/261/343, 143/233/261 , 143/261 , 151/154, 151 /154/213/343, 151/154/343, 151/213, 151 /213/233, 213/233/343, and 233/238/343 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4984.
124. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 5196 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 1 17, 1 17/1 18/143/168/169/392, 1 17/1 18/143/392, 1 17/1 18/168/169, 1 17/1 18/168/169/392, 1 17/143/168/169/170/392, 1 17/143/168/169/392, 1 17/168/169, 1 17/168/169/392, 1 17/168/392, 1 17/169, 1 17/392, 1 18/143, 1 18/143/168/169, 1 18/143/169, 1 18/143/392, 1 18/168, 1 18/168/392, 1 18/169/392, 1 18/392, 143/168/169/392, 143/169, 143/392, 168/169, 168/169/392, 168/392, 169/392, and 392 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5196.
125. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 5286 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 134, 134/182/194/294, 134/182/277/293, 134/182/293/320, 134/182/294, 134/194/277, 134/194/277/317, 134/194/293, 134/194/293/294, 134/194/293/317, 134/194/294, 134/194/317, 134/194/317/320, 134/317, 182, 182/194, 182/194/293/320, 182/293/294, 194, 194/277/293/317, 194/277/317/320, 194/277/320, 194/293/294/317, 194/293/320, 277, 293, 317, 317/320, and 320 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5286.
126. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 5412 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 12/134, 13/134, 13/134/210, 22/134/152, 22/134/235/344, 64/93/134/235, 64/134/344/347, 77/134, 79/134/210, 84/134, 86/134, 87/134, 89/134, 93/134/152/235/344, 93/134/235/344, 93/134/244/344, 93/134/344, 131 , 134/152/344, 134/175, 134/210/382, 134/210/402, 134/210/456, 134/210/478, 134/344, 134/377, 134/378, 134/380, 134/381 , 134/382, 134/384, 134/386, 134/387, 134/388, 134/390, 134/391 , 134/393, 134/395, 134/398, 134/401 , 134/402, 134/406, 134/407, 134/421 , 134/456, 134/457, 134/459, 134/460, 134/464, 134/467, 134/468, 134/470, 134/471 , 134/473, 134/473/474, 134/474, and 134/475 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5412.
127. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 5412 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 73/127/134/160/172/198/290, 73/127/134/160/198/333, 73/127/134/160/210/266,
73/127/134/172/192/266/333, 73/127/134/172/198, 73/127/134/210/290, 73/127/134/210/333, 73/127/134/290/333, 73/127/134/333, 73/134, 73/134/160/172/192/290, 73/134/160/172/198/333, 73/134/160/172/290, 73/134/160/266, 73/134/160/333, 73/134/172, 73/134/172/210/333, 73/134/172/266/333, 73/134/198/210, 73/134/198/210/266, 73/134/210/333, 73/134/333, 73/134/366, 127/134, 127/134/160/172/192/333, 127/134/160/172/210/266/290, 127/134/172/198/266/333, 127/134/192/210, 127/134/198/210/290, 127/134/210/266/290, 127/134/210/333, 134, 134/160/172/333, 134/160/198/266/290, 134/172/192/290/333, 134/192/198, 134/198, 134/198/266/333, 134/210, 134/210/290, and 134/333 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5412.
128. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 5422 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 13, 13/178/381 , 13/178/381/473, 13/178/457, 13/381 , 13/381/457, 13/402/457, 13/457, 178, 178/381/457, 178/381/457/473, 178/402/457, 178/473, 381 , 381/393/457, 381/457/473, 381/473, and 393/457 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5422.
129. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 972 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from
38/73/113/115/117/118/127/134/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/29 5/317/333/392/423, 38/73/113/115/117/118/127/134/152/154/160/163/164/168/169/172/184/192/194/198/220/243/260/266/27 0/295/317/333/392/423,
38/73/113/115/117/118/127/134/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/29 5/317/333/392/423/457,
38/73/113/115/117/118/127/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/295/31 7/333/392/423,
38/73/113/115/118/127/152/154/160/162/164/168/172/184/192/198/220/243/260/266/270/295/317/333/42 3,
38/73/113/115/118/127/152/154/160/163/164/168/169/172/184/192/198/220/243/260/266/270/295/317/33 3/423,
38/73/113/115/118/127/152/154/160/164/168/172/184/189/192/198/220/243/260/266/270/295/317/333/42 3,
38/73/113/115/118/127/152/154/160/164/168/172/184/192/198/210/220/243/260/266/270/295/317/333/42 3, 38/73/113/115/118/127/152/154/160/164/168/172/184/192/198/220/243/260/266/270/295/317/333/423, 38/73/113/118/127/152/154/160/164/168/172/184/192/198/220/243/260/266/270/295/317/333/423, 38/73/113/118/127/152/154/160/164/168/172/184/192/198/220/243/266/270/317/333/423, 38/73/113/118/127/152/154/160/164/168/172/184/192/198/220/266/270/317/333/423, and
38/113/127/152/154/160/164/168/172/198/220/290/317/423 and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 972.
130. The method of any one of Claims 75-80, wherein the reference sequence is SEQ ID NO. 5704 and the at least one substitution or substitution set comprises substitutions at amino acid positions selected from 48, 116, 118, 120, 130, 137, 153, 157, 163, 175, 181 , 187, 192, 213, 246, 263, 273, 296, 318, 334, 341 , 394, 412, 424, 454, and 458, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 5704.
131 . The method of any of Claims 75-130, wherein the engineered transposase comprises a polypeptide sequence selected from the even-numbered sequences selected from SEQ ID NOs: 2-2368, 2388-5694, 5702, and 5706-5756.
132. The method of any one of Claims 75-80 and 131 , wherein the engineered transposase comprises a polypeptide sequence of SEQ ID NO: 2396.
133. The method of any one of Claims 75-80 and 131 , wherein the engineered transposase comprises a polypeptide sequence of SEQ ID NO: 4642.
134. The method of any one of Claims 75-80 and 131 , wherein the engineered transposase comprises a polypeptide sequence of SEQ ID NO: 4872.
135. The method of any one of Claims 75-80 and 131 , wherein the engineered transposase comprises a polypeptide sequence of SEQ ID NO: 4954.
136. The method of any one of Claims 75-80 and 131 , wherein the engineered transposase comprises a polypeptide sequence of SEQ ID NO: 5186.
137. The method of any one of Claims 75-80 and 131 , wherein the engineered transposase comprises a polypeptide sequence of SEQ ID NO: 5354.
138. The method of any one of Claims 75-80 and 131 , wherein the engineered transposase comprises a polypeptide sequence of SEQ ID NO: 5500.
139. The method of any one of Claims 75-80 and 131 , wherein the engineered transposase comprises a polypeptide sequence of SEQ ID NO: 5520.
140. The method of any one of Claims 75-80 and 131 , wherein the engineered transposase comprises a polypeptide sequence of SEQ ID NO: 5702.
141 . The method of any one of Claims 75-80 and 131 , wherein the engineered transposase comprises a polypeptide sequence of SEQ ID NO: 5738.
142. The method of any one of Claims 75-141 , wherein the engineered transposase is purified.
143. The method of any one of Claims 76-141 , wherein fragmenting the target nucleic acid comprises tagmentation or random sheering and adapter ligation.
144. The method of any one of Claims 76-141 , wherein fragmenting the target nucleic acid comprises tagmentation.
145. The method of Claim 75, wherein each altered nucleic acid of the mixture of altered nucleic acids comprises an identifiable sequence tag (1ST).
146. The method of any one of Claims 75-79, wherein each nucleic acid fragment of the subset of nucleic acid fragments comprises an identifiable sequence tag (1ST).
147. The method of Claim 155 or 156, wherein the 1ST is between 6 and 30 nucleotides in length.
148. The method of Claim 75, wherein each altered nucleic acid of the mixture of altered nucleic acids is between 30 and 30000 nucleotides in length.
149. The method of any one of Claims 76-79, wherein each nucleic acid fragment of the plurality of nucleic acid fragments is between 30 and 30000 nucleotides in length.
150. The method of any one of Claims 75-134, wherein the transposition event occurs in reaction conditions additionally comprising a terminal deoxynucleotide transferase, dNTP, and/or buffer components suitable for the addition of deoxynucleotides to the 3’ terminus of each of the plurality of nucleic acid fragments.
151 . The method of any one of Claims 75-144, wherein the transposition event occurs in reaction conditions additionally comprising a DNA ligase and/or buffer components suitable for a ligation reaction.
152. The method of Claim 75, wherein each altered nucleic acid of the mixture of altered nucleic acids further comprises a sample tag and/or a unique molecular identifier (UMI).
153. The method of any one of Claims 76-79, wherein each nucleic acid fragment of the subset of nucleic acid fragments further comprises a sample tag and/or a unique molecular identifier (UMI).
154. The method of any one of Claims 77-144, wherein amplification of the subset of nucleic acid fragments is performed through polymerase chain reaction (PCR), multiple displacement amplification (MDA), ligase chain reaction (LCR), loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), or strand displacement amplification (SDA).
155. The method of any one of Claims 78-144, wherein the sequencing comprises nextgeneration sequencing (NGS).
156. The method of any one of Claims 78-144, wherein sequencing comprises sequencing by synthesis, sequencing by ligation, or nanopore sequencing.
157. The method of Claim 156, wherein the sequencing by synthesis comprises Illumina™ dye sequencing, single-molecule real-time (SMRT™) sequencing, or pyrosequencing.
158. The method of Claim 156, wherein the sequencing by ligation comprises polony-based sequencing or SOLiD™ sequencing.
159. The method of any one of Claims 75-158, wherein the target nucleic acid comprises genomic DNA or cDNAs from a single cell.
160. The method of any one of Claims 75-158, wherein the target nucleic acid comprises nucleic acids from a plurality of haplotypes.
161 . The method of any one of Claims 75-158, wherein the target nucleic acid is crosslinked via histones or chromatin from single or multiple cells.
162. The method of any one of Claims 75-158, wherein the target nucleic acid has been condensed or optionally treated with one or more condensing agents.
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Citations (3)

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US20190169602A1 (en) * 2016-01-12 2019-06-06 Seqwell, Inc. Compositions and methods for sequencing nucleic acids
US20200087635A1 (en) * 2009-02-26 2020-03-19 Poseida Therapeutics, Inc. Hyperactive piggybac transposases
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US20200087635A1 (en) * 2009-02-26 2020-03-19 Poseida Therapeutics, Inc. Hyperactive piggybac transposases
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