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WO2025054409A1 - Transposase tcbuster mutante à solubilité améliorée - Google Patents

Transposase tcbuster mutante à solubilité améliorée Download PDF

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WO2025054409A1
WO2025054409A1 PCT/US2024/045520 US2024045520W WO2025054409A1 WO 2025054409 A1 WO2025054409 A1 WO 2025054409A1 US 2024045520 W US2024045520 W US 2024045520W WO 2025054409 A1 WO2025054409 A1 WO 2025054409A1
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transposase
amino acid
seq
cell
sequence
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Neil OTTO
David NEDRUD
Bryan Jones
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Bio Techne Corp
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Bio Techne Corp
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding

Definitions

  • Transposable genetic elements also called transposons, are segments of DNA that can be mobilized from one genomic location to another within a single cell. Transposons can be divided into two major groups according to their mechanism of transposition: transposition can occur (1) via reverse transcription of an RNA intermediate for elements termed retrotransposons, and (2) via direct transposition of DNA flanked by terminal inverted repeats (TIRs) for DNA transposons. Active transposons encode one or more proteins that are required for transposition. The natural active DNA transposons harbor a transposase enzyme gene.
  • DNA transposons in the hAT family are widespread in plants and animals.
  • a number of active hAT transposon systems have been identified and found to be functional, including but not limited to, the Hermes transposon, Ac transposon, hobo transposon, and the Tol2 transposon.
  • the hAT family is composed of two families that have been classified as the AC subfamily and the Buster subfamily, based on the primary sequence of their transposases.
  • Members of the hAT family belong to Class II transposable elements. Class II mobile elements use a cut and paste mechanism of transposition.
  • hAT elements share similar transposases, short terminal inverted repeats, and an eight base-pairs duplication of genomic target.
  • TcBuster is a member of the hAT family of DNA transposons. TcBuster can be used to stably integrate large gene cassettes into immune or stem cells, such as for immunoncology or regenerative medicine. Use of a transposase as a recombinant protein would shorten editing time and thus minimize safety concerns for editing cells such as transposon hoping and undesirable transposition spots. However, when expressed in E. coli, TcBuster and other transposases are not soluble enough for scalable purification, and thus recombinant transposases are currently not commercially available.
  • a mutant TcBuster transposase comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 1 ; and at least one N-terminal amino acid deletion.
  • the at least one N-terminal amino acid deletion is between amino acid 2 and amino acid and amino acid 48 of SEQ ID NO: 1.
  • the at least one N-terminal amino acid deletion comprises at least two amino acids.
  • the at least one N-terminal amino acid deletion comprises 10 to 48 amino acids.
  • the at least one N-terminal amino acid deletion comprises 20 to 48 amino acids.
  • the at least one N-terminal amino acid deletion comprises 30 to 48 amino acids.
  • the at least one N-terminal amino acid deletion comprises 40 to 48 amino acids. In some embodiments, the at least two N-terminal amino acid deletions are contiguous. In some embodiments, at least two N-terminal amino acid deletions are noncontiguous.
  • the at least one N-terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 48 of SEQ ID NO: 1. In some embodiments, the at least one N- terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 47 of SEQ ID NO: 1. In some embodiments, the at least one N-terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 46 of SEQ ID NO: 1. In some embodiments, the at least one N- terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 45 of SEQ ID NO: 1.
  • the at least one N-terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 36 of SEQ ID NO: 1. In some embodiments, the at least one N- terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 35 of SEQ ID NO: 1.
  • the eukaryotic cell is selected from the group consisting of a CHO cell, a HEK293 cell, an SIP cell, a Saccharomyces cerevisiae cell, and a Pichia pastoris cell, and any derivatives or variants thereof.
  • the transposase is functional.
  • the transposase comprises at least one amino acid substitution relative to SEQ ID NO: 1.
  • the at least one amino acid substitution comprises N85S, D99A, E247K, V377T, and/or E469K with reference to SEQ ID NO: 1.
  • the at least one amino acid substitution comprises two of the following amino acid substitutions with reference to SEQ ID NO: 1: N85S, D99A, E247K, V377T, and E469K.
  • the at least one amino acid substitution comprises three of the following amino acid substitutions with reference to SEQ ID NO: 1: N85S, D99A, E247K, V377T, and E469K.
  • the at least one amino acid substitution comprises four of the following amino acid substitutions with reference to SEQ ID NO: 1 : N85S, D99A, E247K, V377T, and E469K. In some embodiments, the at least one amino acid substitution comprises the following amino acid substitutions with reference to SEQ ID NO: 1 : N85S, D99A, E247K, V377T, and E469K. In some embodiments, the at least one amino acid substitution comprises N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and/or C512E with reference to SEQ ID NO: 1.
  • the at least one amino acid substitution comprises two of the following amino acid substitutions with reference to SEQ ID NO: 1 : N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E. In some embodiments, the at least one amino acid substitution comprises three of the following amino acid substitutions with reference to SEQ ID NO: 1 : N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E.
  • the at least one amino acid substitution comprises four of the following amino acid substitutions with reference to SEQ ID NO: 1 : N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E. In some embodiments, the at least one amino acid substitution comprises five of the following amino acid substitutions with reference to SEQ ID NO: 1 : N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E.
  • the at least one amino acid substitution comprises six of the following amino acid substitutions with reference to SEQ ID NO: 1 : N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E. In some embodiments, the at least one amino acid substitution comprises seven of the following amino acid substitutions with reference to SEQ ID NO: 1 : N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E.
  • the at least one amino acid substitution comprises eight of the following amino acid substitutions with reference to SEQ ID NO: 1 : N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E.
  • the at least one amino acid substitution comprises the following amino acid substitutions with reference to SEQ ID NO: 1 : N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E.
  • the transposase has increased transposition efficiency compared to a wild-type TcBuster transposase having amino acid sequence SEQ ID NO: 1.
  • the transposase is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.
  • the transposase is fused to a DNA sequence specific binding domain.
  • the DNA sequence specific binding domain comprises a TALE domain, a zinc finger domain, an AAV Rep DNA-binding domain, or any combination thereof.
  • the DNA sequence specific binding domain comprises a CRISPR- associated protein.
  • the transposase is fused to a DNA sequence specific binding domain via a linker.
  • the linker is from 3 to 50 amino acids in length.
  • the linker comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 50 amino acids.
  • the linker comprises GGSGGSGGSGGSGTS (SEQ ID NO: 9) or GGGGS (SEQ ID NO: 11).
  • the transposase is fused to a reporter protein.
  • the reporter protein is selected from the group consisting of GFP, RFP, mCherry, YFP, BFP, P-galactosidase (P-gal), alkaline phosphatase (AP), chloramphenicol acetyl transferase (CAT), and horseradish peroxidase (HRP), and any derivatives or variants thereof.
  • the transposase is fused to an affinity tag.
  • the affinity tag is selected from the group consisting of polyhistidine, Glutathione S -Transferase (GST), Maltose Binding Protein (MBP), Calmodulin Binding Peptide (CBP), intein-chitin binding domain (intein-CBD), Streptavidin/Biotin, FLAG, hemagglutinin (HA), c- myc, T7, Glu-Glu, and any derivatives or variants thereof.
  • the transposase is fused to a tag that enhances protein solubility.
  • the tag is selected from the group consisting of Bl domain of Streptococcal protein G (GB-1), Thioredoxin A, Glutathione S- Transferase (GST), Maltose Binding Protein (MBP), Small Ubiquitin-like Modifyer (SUMO), NusA , and any derivatives or variants thereof.
  • polynucleotide that codes for the mutant TcBuster transposase as described herein.
  • polynucleotide that codes for the fusion transposase as described herein.
  • the polynucleotide comprises DNA that encodes the mutant TcBuster transposase or the fusion transposase.
  • the polynucleotide comprises messenger RNA (mRNA) that encodes the mutant TcBuster transposase or the fusion transposase.
  • mRNA messenger RNA
  • the mRNA is chemically modified.
  • the mRNA comprises a 5’ cap.
  • an mRNA cap prevents mRNA from decay during transcription.
  • the 5’ cap comprises a guanosine.
  • the polynucleotide comprises nucleic acid sequence encoding for a transposon recognizable by the mutant TcBuster transposase or the fusion transposase.
  • the polynucleotide is present in a DNA vector.
  • the DNA vector comprises a mini-circle plasmid.
  • the introducing comprises contacting the cell with a polynucleotide encoding the mutant TcBuster transposase or the fusion transposase.
  • the polynucleotide comprises DNA that encodes the mutant TcBuster transposase or the fusion transposase.
  • the polynucleotide comprises messenger RNA (mRNA) that encodes the mutant TcBuster transposase or the fusion transposase.
  • mRNA messenger RNA
  • the mRNA is chemically modified.
  • the mRNA comprises a 5’ cap.
  • an mRNA cap prevents mRNA from decay during transcription.
  • the introducing comprises contacting the cell with a DNA vector that contains the transposon.
  • the DNA vector comprises a mini -circle plasmid.
  • the introducing comprises contacting the cell with a plasmid vector that contains both the transposon and the polynucleotide encoding the mutant TcBuster transposase or the fusion transposase.
  • the introducing comprises contacting the cell with the mutant TcBuster transposase or the fusion transposase as a purified protein.
  • the transposon comprises a cargo cassette positioned between two inverted repeats.
  • a left inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 3.
  • a left inverted repeat of the two inverted repeats comprises SEQ ID NO: 3.
  • a right inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 4.
  • a right inverted repeat of the two inverted repeats comprises SEQ ID NO: 4.
  • a left inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 5. In some embodiments, a left inverted repeat of the two inverted repeats comprises SEQ ID NO: 5. In some embodiments, a right inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 6. In some embodiments, a right inverted repeat of the two inverted repeats comprises SEQ ID NO: 6.
  • a left inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 7. In some embodiments, a left inverted repeat of the two inverted repeats comprises SEQ ID NO: 7. In some embodiments, a right inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 8. In some embodiments, a right inverted repeat of the two inverted repeats comprises SEQ ID NO: 8.
  • the cargo cassette comprises a promoter selected from the group consisting of: CMV, EFS, MND, EFl ⁇ , CAGCs, PGK, UBC, U6, Hl, MND, and Cumate.
  • the cargo cassette comprises a CMV promoter.
  • the cargo cassette is present in a forward direction.
  • the cargo cassette is present in a reverse direction.
  • the cargo cassette comprises a transgene.
  • the transgene codes for a protein selected from the group consisting of: a cellular receptor, an immunological checkpoint protein, a cytokine, and any combination thereof.
  • the transgene codes for a cellular receptor selected from the group consisting of: a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof.
  • the introducing comprises transfecting the cell with the aid of electroporation, microinjection, calcium phosphate precipitation, cationic polymers, dendrimers, liposome, microprojectile bombardment, fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, nucleof ection, or any combination thereof.
  • the introducing comprises electroporating the cell.
  • the cell is a primary cell isolated from a subject.
  • the subject is a human.
  • the subject is a patient with a disease.
  • the subject has been diagnosed with cancer or tumor.
  • the cell is isolated from blood of the subject.
  • the cell comprises a primary immune cell.
  • the cell comprises a primary leukocyte.
  • the cell comprises a primary T cell.
  • the primary T cell comprises a gamma delta T cell, a helper T cell, a memory T cell, a natural killer T cell, an effector T cell, or any combination thereof.
  • the primary immune cell comprises a CD3+ cell.
  • the cell comprises a stem cell.
  • the stem cell is selected from the group consisting of: embryonic stem cell, hematopoietic stem cell, epidermal stem cell, epithelial stem cell, bronchoalveolar stem cell, mammary stem cell, mesenchymal stem cell, intestine stem cell, endothelial stem cell, neural stem cell, olfactory adult stem cell, neural crest stem cell, testicular cell, and any combination thereof.
  • the stem cell comprises induced pluripotent stem cell.
  • Yet another aspect of the present disclosure provides a method of treatment, comprising: (a) introducing into a cell a transposon and the mutant TcBuster transposase or the fusion transposase as described herein, which recognize the transposon, thereby generating a genetically modified cell; (b) administering the genetically modified cell to a patient in need of the treatment.
  • the genetically modified cell comprises a transgene introduced by the transposon.
  • the patient has been diagnosed with cancer or tumor.
  • the administering comprises transfusing the genetically modified cell into blood vessels of the patient.
  • Yet another aspect of the present disclosure provides a system for genome editing, comprising: the mutant TcBuster transposase or fusion transposase as described herein, and a transposon recognizable by the mutant TcBuster transposase or the fusion transposase.
  • Yet another aspect of the present disclosure provides a system for genome editing, comprising: the polynucleotide encoding a mutant TcBuster transposase or fusion transposase as described herein, and a transposon recognizable by the mutant TcBuster transposase or the fusion transposase.
  • the polynucleotide comprises DNA that encodes the mutant TcBuster transposase or the fusion transposase.
  • the polynucleotide comprises messenger RNA (mRNA) that encodes the mutant TcBuster transposase or the fusion transposase.
  • the mRNA is chemically modified.
  • the transposon is present in a DNA vector.
  • the DNA vector comprises a mini -circle plasmid.
  • the polynucleotide and the transposon are present in a same plasmid.
  • the transposon comprises a cargo cassette positioned between two inverted repeats.
  • a left inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 3.
  • a left inverted repeat of the two inverted repeats comprises SEQ ID NO: 3.
  • a right inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 4.
  • a right inverted repeat of the two inverted repeats comprises SEQ ID NO: 4.
  • a left inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 5. In some embodiments, a left inverted repeat of the two inverted repeats comprises SEQ ID NO: 5. In some embodiments, a right inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 6. In some embodiments, a right inverted repeat of the two inverted repeats comprises SEQ ID NO: 6.
  • a left inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 7. In some embodiments, a left inverted repeat of the two inverted repeats comprises SEQ ID NO: 7. In some embodiments, a right inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 8. In some embodiments, a right inverted repeat of the two inverted repeats comprises SEQ ID NO: 8.
  • the cargo cassette comprises a promoter selected from the group consisting of: CMV, EFS, MND, EFl ⁇ , CAGCs, PGK, UBC, U6, Hl, MND, and Cumate.
  • the cargo cassette comprises a CMV promoter.
  • the cargo cassette comprises a transgene.
  • the transgene codes for a protein selected from the group consisting of: a cellular receptor, an immunological checkpoint protein, a cytokine, and any combination thereof.
  • the transgene codes for a cellular receptor selected from the group consisting of: a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof.
  • TCR T cell receptor
  • BCR B cell receptor
  • CAR chimeric antigen receptor
  • the cargo cassette is present in a forward direction. In some embodiments, the cargo cassette is present in a reverse direction.
  • FIG. 1 depicts the amino acid sequence alignment of TcBuster transposase versus a number of transposases in AC subfamily, with only regions of amino acid conservation being shown.
  • a wild-type TcBuster transposase can be regarded as comprising, from N terminus to C terminus, a ZnF-BED domain (amino acids 76-98), a DNA Binding and Oligomerization domain (amino acids 112-213), a first Catalytic domain (amino acids 213-312), an Insertion domain (amino acids 312-543), and a second Catalytic domain (amino acids 583-620), as well as at least four inter-domain regions in between these annotated domains.
  • FIG. 2 shows amino acid sequence of wild-type TcBuster transposase with certain amino acids annotated (SEQ ID NO: 1).
  • FIG. 3 shows the predicted structure of TcBuster using Alphafold.
  • FIG. 4 shows multiple sequence alignment of the first 50 amino acids of TcBuster With Other HAT Family Transposases.
  • FIG. 5 shows the putative NLS for TcBuster.
  • the putative NLS GTTSRKKRKYD (SEQ ID NO: 25) was strongly predicted.
  • Other putative NLS were with lower scores were TSRKKRKYDE (SEQ ID NO: 26), SRKKRKYDED (SEQ ID NO: 27), and EYFKRKCNEL (SEQ ID NO: 28).
  • FIG. 6 shows that truncated version of TcBuster are more soluble than full-length TcBuster.
  • FIG. 7 shows that the increased solubility of truncated TcBuster proteins as shown in FIG. 5 is consistent across a variety of E. Coli strains.
  • FIG. 8 shows that increased solubility of truncated TcBuster proteins (e.g. as shown in FIG. 6 and FIG. 7) is consistent across a variety of experiments.
  • FIG. 9A, FIG. 9B, and FIG. 9C show that truncated TcBuster variants are as active as full length TcBuster and provides higher expressing populations of cells earlier than full length.
  • DNA transposons can translocate via a non-replicative, ‘cut-and-paste’ mechanism. This requires recognition of the two terminal inverted repeats by a catalytic enzyme, i.e. transposase, which can cleave its target and consequently release the DNA transposon from its donor template. Upon excision, the DNA transposons may subsequently integrate into the acceptor DNA that is cleaved by the same transposase. In some of their natural configurations, DNA transposons are flanked by two inverted repeats and may contain a gene encoding a transposase that catalyzes transposition.
  • transposon For genome editing applications with DNA transposons, it is desirable to design a transposon to develop a binary system based on two distinct plasmids whereby the transposase is physically separated from the transposon DNA containing the gene of interest flanked by the inverted repeats. Co-delivery of the transposon and transposase plasmids into the target cells enables transposition via a conventional cut-and-paste mechanism.
  • TcBuster is a member of the hAT family of DNA transposons. Other members of the family include Sleeping Beauty and PiggBac. Tranposases are typically unstable and insoluble proteins, and currently there are no commercially available transposases that are recombinant proteins.
  • the present disclosure relates to mutant TcBuster transposases comprising at least one N-terminal amino acid deletion.
  • the mutant TcBuster transposases provided herein are soluble when expressed in a host cell, such as a bacterial cell. Accordingly, the mutant TcBuster transposases provided herein are advantageous over other transposases in that they are sufficiently soluble in a host cell and thus large scale manufacture and isolation is feasible.
  • a mutant TcBuster transposase may comprise one or more amino acid substitutions in comparison to a wild-type TcBuster transposase (SEQ ID NO: 1) and at least one N-terminal amino acid deletion.
  • TcBuster transposase comprises an amino acid sequence having at least 70% sequence identity to full length sequence of a wild-type TcBuster transposase (SEQ ID NO: 1) and at least one N-terminal amino acid deletion.
  • a mutant TcBuster transposase comprises an amino acid sequence having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to full length sequence of a wild-type TcBuster transposase (SEQ ID NO: 1) and at least one N-terminal amino acid deletion.
  • a mutant TcBuster transposase comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to full length sequence of a wild-type TcBuster transposase (SEQ ID NO: 1) and at least one N-terminal amino acid deletion.
  • a mutant TcBuster transposase comprises an amino acid sequence having at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or at least 99.95% sequence identity to full length sequence of a wild-type TcBuster transposase (SEQ ID NO: 1) and at least one N-terminal amino acid deletion.
  • percent (%) identity can refer to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment, for purposes of determining percent identity, can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software.
  • Percent identity of two sequences can be calculated by aligning a test sequence with a comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to amino acids or nucleotides in the same position of the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.
  • complement can refer to a sequence that is fully complementary to and hybridizable to the given sequence.
  • a sequence hybridized with a given nucleic acid is referred to as the “complement” or “reverse-complement” of the given molecule if its sequence of bases over a given region is capable of complementarily binding those of its binding partner, such that, for example, A-T, A-U, G-C, and G-U base pairs are formed.
  • a first sequence that is hybridizable to a second sequence is specifically or selectively hybridizable to the second sequence, such that hybridization to the second sequence or set of second sequences is preferred (e.g. thermodynamically more stable under a given set of conditions, such as stringent conditions commonly used in the art) to hybridization with non-target sequences during a hybridization reaction.
  • hybridizable sequences share a degree of sequence complementarity over all or a portion of their respective lengths, such as between 25%-100% complementarity, including at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence complementarity.
  • Sequence identity such as for the purpose of assessing percent complementarity, can be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see e.g.
  • the EMBOSS Needle aligner available at ebi.ac.uk/Tools/psa/emboss_needle/ nucleotide.html optionally with default settings
  • the BLAST algorithm see e.g. the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with default settings
  • the Smith- Waterman algorithm see e.g. the EMBOSS Water aligner available at ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html, optionally with default settings.
  • Optimal alignment can be assessed using any suitable parameters of a chosen algorithm, including default parameters.
  • Complementarity can be perfect or substantial/sufficient. Perfect complementarity between two nucleic acids can mean that the two nucleic acids can form a duplex in which every base in the duplex is bonded to a complementary base by Watson-Crick pairing. Substantial or sufficient complementary can mean that a sequence in one strand is not completely and/or perfectly complementary to a sequence in an opposing strand, but that sufficient bonding occurs between bases on the two strands to form a stable hybrid complex in set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequences and standard mathematical calculations to predict the Tm of hybridized strands, or by empirical determination of Tm by using routine methods.
  • hybridization conditions e.g., salt concentration and temperature
  • mutant TcBuster transposase comprising an amino acid sequence that is at least 70% identical (e.g. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 1 and at least one N-terminal amino acid deletion.
  • the mutant TcBuster transposase is functional.
  • the term “functional” indicates that the mutant TcBuster transposase displays transposase activity (e.g.
  • a “functional” mutant TcBuster provided herein comprises at least one N-terminal amino acid deletion which imparts improved solubility without substantially disrupting transposase activity of the mutant TcBuster transposase.
  • the at least one N-terminal amino acid deletion is between amino acid 2 and amino acid and amino acid 48 of SEQ ID NO: 1. In some embodiments, the at least one N-terminal amino acid deletion comprises at least two amino acids. In some embodiments, the at least one N-terminal amino acid deletion comprises 10 to 48 amino acids. In some embodiments, the at least one N-terminal amino acid deletion comprises 20 to 48 amino acids. In some embodiments, the at least one N-terminal amino acid deletion comprises 30 to 48 amino acids. In some embodiments, the least one N-terminal amino acid deletion comprises 40 to 48 amino acids.
  • the at least one N-terminal amino acid deletion comprises a deletion of 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids between amino acid 2 and amino acid 48 of SEQ ID NO: 1.
  • the mutant TcBuster transposase comprises at least two N-terminal amino acid deletions.
  • the at least two N-terminal amino acid deletion are contiguous.
  • the at least two N-terminal amino acid deletion are non-contiguous.
  • the at least one N-terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 48 of SEQ ID NO: 1. In some embodiments, the at least one N- terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 47 of SEQ ID NO: 1. In some embodiments, the at least one N-terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 46 of SEQ ID NO: 1. In some embodiments, the at least one N- terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 45 of SEQ ID NO: 1.
  • the at least one N-terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 44 of SEQ ID NO: 1. In some embodiments, the at least one N- terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 43 of SEQ ID NO: I. In some embodiments, the at least one N-terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 42 of SEQ ID NO: 1 . In some embodiments, the at least one N- terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 41 of SEQ ID NO: 1.
  • the at least one N-terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 40 of SEQ ID NO: 1. In some embodiments, the at least one N- terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 39 of SEQ ID NO: 1. In some embodiments, the at least one N-terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 38 of SEQ ID NO: 1. In some embodiments, the at least one N- terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 37 of SEQ ID NO: 1.
  • the at least one N-terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 36 of SEQ ID NO: 1. In some embodiments, the at least one N- terminal amino acid deletion is an N-terminal truncation of amino acids 2 to 35 of SEQ ID NO: 1.
  • the mutant TcBuster transposase is soluble when expressed in a host cell.
  • soluble refers to a protein that is present in a correctly folded structure within the cytoplasm of a host cell.
  • insoluble proteins are present in a denatured form within cytoplasmic granules (i.e. inclusion bodies) within the host cell.
  • the mutant TcBuster transposase is soluble when expressed in a bacterial host cell.
  • the host cell is a bacterial cell is from an E. coli strain. Suitable E.
  • the mutant TcBuster transposase is soluble when expressed in a eukaryotic host cell.
  • eukaryotic cells include, for example, CHO cells, HEK293 cells, Sf9 cells, Saccharomyces cerevisiae cells, Pichia pastoris cells, and any derivatives or variants thereof.
  • a wild-type TcBuster transposase can be regarded as comprising, from N terminus to C terminus, a ZnF-BED domain (amino acids 76-98), a DNA Binding and Oligomerization domain (amino acids 112-213), a first Catalytic domain (amino acids 213-312), an Insertion domain (amino acids 312-543), and a second Catalytic domain (amino acids 583-620), as well as at least four inter-domain regions in between these annotated domains.
  • numerical references to amino acids, as used herein, are all in accordance to SEQ ID NO: 1.
  • a mutant TcBuster transposase can comprise one or more amino acid substitutions in any one of these domains, or any combination thereof, in addition to the at least one N-terminal amino acid deletion.
  • Exemplary mutations in comparison to a wildtype TcBuster transposase (SEQ ID NO: 1) are described in PCT/US2017/066829 and PCT/US2019/038410, the entire contents of each of which are incorporated herein by reference for all purposes.
  • a mutant TcBuster transposase can comprise one or more amino acid substitutions in ZnF-BED domain, a DNA Binding and Oligomerization domain, a first Catalytic domain, an Insertion domain, or a combination thereof, in addition to the at least one N-terminal amino acid deletion.
  • a mutant TcBuster transposase can comprise one or more amino acid substitutions in at least one of the two catalytic domains.
  • An exemplary mutant TcBuster transposase can comprise one or more amino acid substitutions from Table 1 or Table 1.1, in addition to the at least one N-terminal amino acid deletion.
  • a mutant TcBuster transposase can comprise at least one of the amino acid substitutions from Table 1 or Table 1.1.
  • a mutant TcBuster transposase can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, or more of the amino acid substitutions from Table 1 or Table 1.1, in addition to the at least one N-terminal amino acid deletion.
  • An exemplary mutant TcBuster transposase comprises one or more amino acid substitutions, or combinations of substitutions, from Table 2, in addition to the at least one N- terminal amino acid deletion.
  • a mutant TcBuster transposase can comprise at least one of the amino acid substitutions, or combinations of substitutions, from Table 2, in addition to the at least one N-terminal amino acid deletion.
  • a mutant TcBuster transposase can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, or more of the amino acid substitutions, or combinations of substitutions, from Table 2, in addition to the at least one N-terminal amino acid deletion.
  • An exemplary mutant TcBuster transposase comprises one or more amino acid substitutions, or combinations of substitutions, from Table 3, in addition to the at least one N- terminal amino acid deletion.
  • a mutant TcBuster transposase can comprise at least one of the amino acid substitutions, or combinations of substitutions, from Table 3, in addition to the at least one N-terminal amino acid deletion.
  • a mutant TcBuster transposase can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, or more of the amino acid substitutions, or combinations of substitutions, from Table 3, in addition to the at least one N-terminal amino acid deletion.
  • the mutant TcBuster transposase comprises at least one amino acid substitution relative to SEQ ID NO: 1, and at least one N-terminal amino acid deletion (e.g. a deletion of one or more amino acids, such as a deletion of 10-48 amino acids, 20-48 amino acids, 30-48 amino acids, or 40-48 amino acids between amino acids 2 and 48 of SEQ ID NO: 1).
  • the at least one amino acid substitution comprises N85S, D99A, E247K, V377T, and/or E469K with reference to SEQ ID NO: 1.
  • the at least one amino acid substitution comprises two of N85S, D99A, E247K, V377T, and E469K with reference to SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution comprises three of N85S, D99A, E247K, V377T, and E469K with reference to SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution comprises four of N85S, D99A, E247K, V377T, and E469K with reference to SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution comprises each of N85S, D99A, E247K, V377T, and E469K with reference to SEQ ID NO: 1.
  • a “hyperactive” mutant TcBuster transposase can refer to any mutant TcBuster transposase that has increased transposition efficiency as compared to a wildtype TcBuster transposase having amino acid sequence SEQ ID NO: 1.
  • a hyperactive mutant TcBuster transposase may have increased transposition efficiency under certain situations as compared to a wild-type TcBuster transposase having amino acid sequence SEQ ID NO: 1, and increased solubility due to the at least one N- terminal amino acid deletion.
  • the hyperactive mutant TcBuster transposase may have better transposition efficiency than the wild-type TcBuster transposase when being used to catalyze transposition of transposons having particular types of inverted repeat sequences.
  • the hyperactive mutant TcBuster transposase does not have increased transposition efficiency in comparison to the wild-type TcBuster transposase.
  • the hyperactive mutant TcBuster transposase may have increased transposition efficiency in comparison to a wild-type TcBuster transposase having amino acid sequence SEQ ID NO: 1, under certain transfection conditions.
  • a hyperactive mutant TcBuster transposase when compared to a wild-type TcBuster transposase, may have better transposition efficiency when the temperature is higher than normal cell culture temperature; a hyperactive mutant TcBuster transposase may have better transposition efficiency in a relative acidic or basic aqueous medium; a hyperactive mutant TcBuster transposase may have better transposition efficiency when a particular type of transfection technique (e.g. electroporation) is performed.
  • Transposition efficiency can be measured by the percent of successful transposition events occurring in a population of host cells normalized by the amount of transposon and transposase introduced into the population of host cells.
  • the same transposon construct is paired with each of the two or more transposases for transfection of the host cells under same or similar transfection conditions.
  • the amount of transposition events in the host cells can be examined by various approaches.
  • the transposon construct may be designed to contain a reporter gene positioned between the inverted repeats, and transfected cells positive for the reporter gene can be counted as the cells where successful transposition events occurs, which can give an estimate of the amount of the transposition events.
  • Another non-limiting example includes sequencing of the host cell genome to examine the insertion of the cassette cargo of the transposon.
  • the same transposase when the transposition efficiency of two or more different transposons is compared, the same transposase can be paired with each of the different transposons for transfection of the host cells under same or similar transfection conditions. Similar approaches can be utilized for the measurement of transposition efficiency. Other methods known to one skilled in the art may also be implemented for the comparison of transposition efficiency.
  • One exemplary method can comprise systemically mutating amino acids of TcBuster transposase to increase a net charge of the amino acid sequence.
  • the method can comprise performing systematic alanine scanning to mutate aspartic acid (D) or glutamic acid (E), which are negatively charged at a neutral pH, to alanine residues.
  • a method can comprise performing systemic mutation to lysing (K) or arginine (R) residues, which are positively charged at a neutral pH.
  • increase in a net charge of the amino acid sequence at a neutral pH may increase the transposition efficiency of the TcBuster transposase.
  • the transposition efficiency is expected to increase.
  • positively charged amino acids can form points of contact with DNA target and allow the catalytic domains to act on the DNA target. It may also be contemplated that loss of these positively charged amino acids can decrease either excision or integration activity in transposases.
  • Fig. 2 depicts the WT TcBuster transposase amino acid sequence, highlighting amino acids that may be points of contact with DNA.
  • large bold lettering indicates catalytic triad amino acids; lettering with boxes indicates amino acids that when substituted to a positive charged amino acid increases transposition; italicized and lowercased lettering indicates positive charged amino acids that when substituted to a different amino acid decreases transposition; bold italicized and underlined indicates amino acids that when substituted to a positive charged amino acid increases transposition, and when substituted to a negative charged amino acid decreases transposition; underlined lettering indicates amino acids that could be positive charged amino acids based on protein sequence alignment to the Buster subfamily.
  • a mutant TcBuster transposase can comprise one or more amino acid substitutions that increase a net charge at a neutral pH in comparison to SEQ ID NO: 1, in addition to the at least one N-terminal amino acid deletion.
  • a mutant TcBuster transposase comprising one or more amino acid substitutions that increase a net charge at a neutral pH in comparison to SEQ ID NO: 1 can be hyperactive.
  • the mutant TcBuster transposase can comprise one or more substitutions to a positively charged amino acid, such as, but not limited to, lysine (K) or arginine (R).
  • a mutant TcBuster transposase can comprise one or more substitutions of a negatively charged amino acid, such as, but not limited to, aspartic acid (D) or glutamic acid (E), with a neutral amino acid, or a positively charged amino acid.
  • a negatively charged amino acid such as, but not limited to, aspartic acid (D) or glutamic acid (E)
  • D aspartic acid
  • E glutamic acid
  • One non-limiting example includes a mutant TcBuster transposase that comprises one or more amino acid substitutions that increase a net charge at a neutral pH within or in proximity to a catalytic domain in comparison to SEQ ID NO: 1.
  • the catalytic domain can be the first catalytic domain or the second catalytic domain.
  • the catalytic domain can also include both catalytic domains of the transposase.
  • An exemplary method of the present disclosure can comprise mutating amino acids that are predicted to be in close proximity to, or to make direct contact with, the DNA. These amino acids can be substituted amino acids identified as being conserved in other member(s) of the hAT family (e.g., other members of the Buster and/or Ac subfamilies).
  • the amino acids predicted to be in close proximity to, or to make direct contact with, the DNA can be identified, for example, by reference to a crystal structure, predicted structures, mutational analysis, functional analysis, alignment with other members of the hAT family, or any other suitable method.
  • TcBuster transposase like other members of the hAT transposase family, has a DDE motif, which may be the active site that catalyzes the movement of the transposon. It is contemplated that D223, D289, and E589 make up the active site, which is a triad of acidic residues.
  • the DDE motif may coordinate divalent metal ions and can be important in the catalytic reaction.
  • a mutant TcBuster transposase can comprise one or more amino acid substitutions that increase a net charge at a neutral pH in comparison to SEQ ID NO: 1, and the one or more amino acids are located in proximity to D223, D289, or E589, when numbered in accordance to SEQ ID NO: 1.
  • a mutant TcBuster transposase as provided herein does not comprise any disruption of the catalytic triad, i.e. D223, D289, or E589.
  • a mutant TcBuster transposase may not comprise any amino acid substitution at D223, D289, or E589.
  • a mutant TcBuster transposase may comprises amino acid substitution at D223, D289, or E589, but such substitution does not disrupt the catalytic activity contributed by the catalytic triad.
  • the term “proximity” can refer to a measurement of a linear distance in the primary structure of the transposase. For instance, the distance between D223 and D289 in the primary structure of a wild-type TcBuster transposase is 66 amino acids. In certain embodiments, the proximity can refer to a distance of about 70 to 80 amino acids. In many cases, the proximity can refer to a distance of about 80, 75, 70, 60, 50, 40, 30, 20, 10, or 5 amino acids.
  • the term “proximity” can refer to a measurement of a spatial relationship in the secondary or tertiary structure of the transposase, i.e. when the transposase folds into its three dimensional configurations.
  • Protein secondary structure can refer to three dimensional form of local segments of proteins. Common secondary structural elements include alpha helices, beta sheets, beta turns and omega loops. Secondary structure elements may form as an intermediate before the protein folds into its three dimensional tertiary structure.
  • Protein tertiary structure can refer to the three dimensional shape of a protein. Protein tertiary structure may exhibit dynamic configurational change under physiological or other conditions.
  • the tertiary structure will have a single polypeptide chain "backbone" with one or more protein secondary structures, the protein domains.
  • Amino acid side chains may interact and bond in a number of ways. The interactions and bonds of side chains within a particular protein determine its tertiary structure.
  • the proximity can refer to a distance of about 1 A, about 2A, about 5A, about 8A, about 10A, about 15A, about 20A, about 25 A, about 30A, about 35A, about 40 A, about 50 A, about 60 A, about 70A, about 80 A, about 90 A, or about 100 A.
  • a neutral pH can be a pH value around 7.
  • a neutral pH can be a pH value between 6.9 and 7.1, between 6.8 and 7.2, between 6.7 and 7.3, between 6.6 and 7.4, between 6.5 and 7.5, between 6.4 and 7.6, between 6.3 and 7.7, between 6.2-7.8, between 6.1-7.9, between 6.0-8.0, between 5-8, or in a range derived therefrom.
  • Non-limiting exemplary mutant TcBuster transposases that comprise one or more amino acid substitutions that increase a net charge at a neutral pH in comparison to SEQ ID NO: 1 include TcBuster transposases comprising at least one of the combinations of amino acid substitutions from Table 4, Table 4.1, or both.
  • a mutant TcBuster transposase can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, or more of the amino acid substitutions from Table 4, Table 4.1, or both, in addition to the at least one N-terminal amino acid deletion.
  • a mutant TcBuster transposase can comprise one or more amino acid substitutions that increase a net charge at a non-neutral pH in comparison to SEQ ID NO: 1, in addition to the at least one N-terminal amino acid deletion.
  • the net charge is increased within or in proximity to a catalytic domain at a non-neutral pH.
  • the net charge is increased in proximity to D223, D289, or E589, at a non-neutral pH.
  • the non- neutral pH can be a pH value lower than 7, lower than 6.5, lower than 6, lower than 5.5, lower than 5, lower than 4.5, lower than 4, lower than 3.5, lower than 3, lower than 2.5, lower than 2, lower than 1.5, or lower than 1.
  • the non-neutral pH can also be a pH value higher than 7, higher than 7.5, higher than 8, higher than 8.5, higher than 9, higher than 9.5, or higher than 10.
  • a method can comprise systemically mutating amino acids in the DNA Binding and Oligomerization domain in addition to deleting one or more amino acids in the N-terminus (e.g. one or more of amino acids 2 to 48 of SEQ ID NO: 1).
  • mutation in the DNA Binding and Oligomerization domain may increase the binding affinity to DNA target and promote oligomerization activity of the transposase, which consequentially may promote transposition efficiency.
  • the method can comprise systemically mutating amino acids one by one within or in proximity to the DNA Binding and Oligomerization domain (e.g., amino acid 112 to 213).
  • the method can also comprise mutating more than one amino acid within or in proximity to the DNA Binding and Oligomerization domain.
  • the method can also comprise mutating one or more amino acids within or in proximity to the DNA Binding and Oligomerization domain, together with one or more amino acids outside the DNA Binding and Oligomerization domain.
  • the method can comprise performing rational replacement of selective amino acid residues based on multiple sequence alignments of TcBuster with other hAT family transposases (Ac, Hermes, Hobo, Tag2, Tam3, Hermes, Restless and Tol2) or with other members of Buster subfamily (e.g., AeBusterl, AeBuster2, AeBuster3, BtBusterl, BtBuster2, CfBusterl, and CfBuster2).
  • Buster subfamily e.g., AeBusterl, AeBuster2, AeBuster3, BtBusterl, BtBuster2, CfBusterl, and CfBuster2
  • conservancy of certain amino acids among other hAT family transposases, especially among the active ones may indicate their importance for the catalytic activity of the transposases.
  • the method may comprise obtaining sequences of TcBuster as well as other hAT family transposases; aligning the sequences and identifying the amino acids in TcBuster transposase with a different conserved counterpart among the other hAT family transposases; performing site-directed mutagenesis to produce mutant TcBuster transposase harboring the mutation(s).
  • a hyperactive mutant TcBuster transposase can comprise one or more amino acid substitutions based on alignment to other members of Buster subfamily or other members of hAT family in addition to the at least one N-terminal amino acid deletion.
  • the one or more amino acid substitutions can be substitutions of conserved amino acid for the unconserved amino acid in wild-type TcBuster sequence (SEQ ID NO: 1).
  • Non-limiting examples of mutant TcBuster transposases include TcBuster transposases comprising at least one of the amino acid substitutions from Table 5, Table 5.1, or both.
  • a mutant TcBuster transposase can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, or more of the amino acid substitutions from Table 5, Table 5.1, or both, in addition to the at least one N-terminal amino acid deletion.
  • Another exemplary method can comprise systemically mutating acidic amino acids to basic amino acids and identifying hyperactive mutant transposase.
  • mutant TcBuster transposase can comprise amino acid substitutions V377T, E469K, and D189A.
  • a mutant TcBuster transposase can comprise amino acid substitutions K573E and E578L.
  • a mutant TcBuster transposase can comprise amino acid substitution I452K.
  • a mutant TcBuster transposase can comprise amino acid substitution A358K.
  • a mutant TcBuster transposase can comprise amino acid substitution V297K.
  • a mutant TcBuster transposase can comprise amino acid substitution N85S.
  • a mutant TcBuster transposase can comprise amino acid substitutions N85S, V377T, E469K, and D189A.
  • a mutant TcBuster transposase can comprise amino acid substitutions I452F, V377T, E469K, and D189A.
  • a mutant TcBuster transposase can comprise amino acid substitutions A358K, V377T, E469K, and D189A.
  • a mutant TcBuster transposase can comprise amino acid substitutions V377T, E469K, D189A, K573E and E578L.
  • the mutant TcBuster transposase comprises at least one amino acid substitution relative to SEQ ID NO: 1, and at least one N-terminal amino acid deletion (e.g. a deletion of one or more amino acids, such as 10-48 amino acids, 20-48 amino acids, 30-48 amino acids, or 40-48 amino acids between amino acids 2 and 48 of SEQ ID NO: 1).
  • the at least one amino acid substitution comprises N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and/or C512E with reference to SEQ ID NO: 1.
  • the at least one amino acid substitution comprises two of N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E with reference to SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution comprises three of N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E with reference to SEQ ID NO: 1.
  • the at least one amino acid substitution comprises four of N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E with reference to SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution comprises five of N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E with reference to SEQ ID NO: 1.
  • the at least one amino acid substitution comprises six of N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E with reference to SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution comprises seven of N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E with reference to SEQ ID NO: 1 .
  • the at least one amino acid substitution comprises eight of N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E with reference to SEQ ID NO: 1.
  • the at least one amino acid substitution comprises N209E, T219S, L268T, Y284I, V356L, C470M, A472P, K490I, and C512E with reference to SEQ ID NO: 1.
  • the fusion transposase can comprise a TcBuster transposase sequence and a DNA sequence specific binding domain.
  • the TcBuster transposase sequence of a fusion transposase can comprise an amino acid sequence of any of the mutant TcBuster transposases as described herein.
  • the TcBuster transposase sequence of a fusion transposase can comprise an amino acid sequence of a mutant TcBuster transposase having at least 70% identity to SEQ ID NO: 1 and at least one N- terminal amino acid deletion.
  • a DNA sequence specific binding domain as described herein can refer to a protein domain that is adapted to bind to a DNA molecule at a sequence region (“target sequence”) containing a specific sequence motif.
  • target sequence a sequence region containing a specific sequence motif.
  • an exemplary DNA sequence specific binding domain may selectively bind to a sequence motif TATA (SEQ ID NO: 17), while another exemplary DNA sequence specific binding domain may selectively bind to a different sequence motif ATGCNTAGAT (SEQ ID NO: 18) (N denotes any one of A, T, G, and C).
  • a fusion transposase as provided herein may direct sequence specific insertion of the transposon.
  • a DNA sequence specific binding domain may guide the fusion transposase to bind to a target sequence based on the binding specificity of the binding domain. Being bound to or restricted to a certain sequence region may spatially limit the interaction between the fusion transposase and the transposon, thereby limiting the catalyzed transposition to a sequence region in proximity to the target sequence.
  • the distance of the actual transposition site to the target sequence may vary. Proper design of the fusion transposase configuration can direct the transposition to a desirable target genomic region.
  • a target genomic region for transposition can be any particular genomic region, depending on application purposes. For instance, sometimes, it is desirable to avoid transcription start sites for the transposition, which may cause undesirable, or even harmful, change in expression level of certain important endogenous gene(s) of the cell.
  • a fusion transposase may contain a DNA sequence specific binding domain that can target the transposition to a safe harbor of the host genome.
  • safe harbors can include HPRT, AAVS site (e.g. AAVS1 , AAVS2, ETC.), CCR5, or Rosa26. Safe harbor sites can generally refer to sites for transgene insertion whose use exert little to none disrupting effects on genome integrity of the cell or cellular health and functions.
  • a DNA sequence specific binding domain may be derived from, or be a variant of any DNA binding protein that has sequence- specificity.
  • a DNA sequence specific binding domain may comprise an amino acid sequence at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a naturally occurring sequence-specific DNA binding protein.
  • a DNA sequence specific binding domain may comprise an amino acid sequence at least 70% identical to a naturally occurring sequence-specific DNA binding protein.
  • Non-limiting examples of a naturally occurring sequence-specific DNA binding protein include, but not limited to, transcription factors from various origins, specific-sequence nucleases, and viral replication proteins.
  • a naturally occurring sequence-specific DNA binding protein can also be any other protein having the specific binding capability from various origins. Selection and prediction of DNA binding proteins can be conducted by various approaches, including, but not limited to, using computational prediction databases available online, like DP-Bind (http://lcg.rit.albany.edu/dp-bind/) or DNABIND (http://dnabind.szialab.org/)
  • transcription factor can refer to a protein that controls the rate of transcription of genetic information from DNA to messenger DNA, by binding to a specific DNA sequence.
  • a transcription factor that can be used in a fusion transposase described herein can be based on a prokaryotic transcription factor or a eukaryotic transcription factor, as long as it confers sequence specificity when binding to the target DNA molecule.
  • Transcription factor prediction databases such as DBD (transcriptionfactor.org) may be used for selection of appropriate transcription factor for application of the disclosure herein.
  • a DNA sequence specific binding domain as used herein can comprise one or more DNA binding domain from a naturally occurring transcription factor.
  • DNA binding domains of transcription factors include DNA binding domains that belong to families like basic helix-loop-helix, basic-leucine zipper (bZIP), C-terminal effector domain of the bipartite response regulators, AP2/ERF/GCC box, helix-turn-helix, homeodomain proteins, lambda repressor-like, srf-like (serum response factor), paired box, winged helix, zinc fingers, multi-domain Cys2His2 (C2H2) zinc fingers, Zn2/Cys6, or Zn2/Cys8 nuclear receptor zinc finger.
  • C2H2 Cys2His2
  • a DNA sequence specific binding domain can be an artificially engineered amino acid sequence that binds to specific DNA sequences.
  • artificially designed amino acid sequence include sequences created based on frameworks like transcription activator like effector nucleases (TALEs) DNA binding domain, zinc finger nucleases, adeno associated virus (AAV) Rep protein, and any other suitable DNA binding proteins as described herein.
  • TALEs transcription activator like effector nucleases
  • AAV adeno associated virus
  • Natural TALEs are proteins secreted by Xanthomonas bacteria to aid the infection of plant species. Natural TALEs can assist infections by binding to specific DNA sequences and activating the expression of host genes. In general, TALE proteins consist of a central repeat domain, which determines the DNA targeting specificity and can be rapidly synthesized de novo. TALEs have a modular DNA-binding domain (DBD) containing repetitive sequences of residues. In some TALEs, each repeat region contains 34 amino acids.
  • DBD DNA-binding domain
  • TALE domain as used herein can refer to the modular DBD of TALEs.
  • a pair of residues at the 12th and 13th position of each repeat region can determine the nucleotide specificity and are referred to as the repeat variable diresidue (RVD).
  • the last repeat region termed the half-repeat, is typically truncated to 20 amino acids. Combining these repeat regions allows synthesizing sequence-specific synthetic TALEs.
  • the C-terminus typically contains a nuclear localization signal (NLS), which directs a TALE to the nucleus, as well as a functional domain that modulates transcription, such as an acidic activation domain (AD).
  • NLS nuclear localization signal
  • AD acidic activation domain
  • the endogenous NLS can be replaced by an organism-specific localization signal.
  • an NLS derived from the simian virus 40 large T-antigen can be used in mammalian cells.
  • a list of RVDs and their binding preferences under certain circumstances for nucleotides can be found in Table 7.
  • Additional TALE RVDs can also be used for custom degenerate TALE-DNA interactions.
  • NA has high affinity for all four bases of DNA.
  • N* where * is an RVD with a deletion in the 13th residue, can accommodate all letters of DNA including methylated cytosine.
  • S* may have the ability to bind to any DNA nucleotide.
  • TALE-NT https://tale-nt.cac.cornell.edu/
  • Mojo hand talendesign.org/
  • kits may also assist in creating custom assembly of TALE repeat regions between the N and C-terminus of the protein. These methods can be used to assemble custom DBDs, which are then cloned into an expression vector containing a functional domain, e.g. TcBuster transposase sequence.
  • TALEs can be synthesized de novo in the laboratory, for example, by combining digestion and ligation steps in a Golden Gate reaction with type II restriction enzymes. Al ternatively, TALE can be assembled by a number of different approaches, including, but not limited to, Ligation-Independent Cloning (LIC), Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) assembly, and Iterative-Capped Assembly (ICA).
  • LIC Ligation-Independent Cloning
  • FLASH Fast Ligation-based Automatable Solid-phase High-throughput
  • ICA Iterative-Capped Assembly
  • Zinc fingers are ⁇ 30 amino acids that can bind to a limited combination of ⁇ 3 nucleotides.
  • the C2H2 ZF domain may be the most common type of ZF and appears to be one of the most abundantly expressed proteins in eukaryotic cells.
  • ZFs are small, functional and independently folded domains coordinated with zinc molecules in their structure. Amino acids in each ZF can have affinity towards specific nucleotides, causing each finger to selectively recognize 3-4 nucleotides of DNA.
  • Multiple ZFs can be arranged into a tandem array and recognize a set of nucleotides on the DNA. By using a combination of different zinc fingers, a unique DNA sequence within the genome can be targeted. Different ZFPs of various lengths can be generated, which may allow for recognition of almost any desired DNA sequence out of the possible 64 triplet subsites.
  • Zinc fingers to be used in connection with the present disclosure can be created using established modular assembly fingers, such as a set of modular assembly finger domains developed by Barbas and colleagues, and also another set of modular assembly finger domains by ToolGen. Both set of domains cover all 3 bp GNN, most ANN, many CNN and some TNN triplets (where N can be any of the four nucleotides). Both have a different set of fingers, which allows for searching and coding different ZF modules as needed.
  • a combinatorial selectionbased oligomerized pool engineering (OPEN) strategy can also be employed to minimize context-dependent effects of modular assembly involving the position of a finger in the protein and the sequence of neighboring fingers.
  • OPEN ZF arrays are publicly available from the Zinc Finger Consortium Database.
  • AAV Rep DNA-binding domain is another DNA sequence specific binding domain that can be used in connection with the subject matter of the present disclosure.
  • Viral cis -acting inverted terminal repeats (ITRs), and the trans-acting viral Rep proteins (Rep) are believed to be the factors mediating preferential integration of AAV into AAVS1 site of the host genome in the absence of a helper virus.
  • AAV Rep protein can bind to specific DNA sequence in the AAVS 1 site. Therefore, a site-specific DNA-binding domain can be fused together with a TcBuster transposase domain as described herein.
  • a fusion transposase as provided herein can comprise a TcBuster transposase sequence and a tag sequence.
  • a tag sequence as provide herein can refer to any protein sequence that can be used as a detection tag of the fusion protein, such as, but not limited to, reporter proteins and affinity tags that can be recognized by antibodies.
  • a tag sequence can also refer to a moiety that enhances protein solubility.
  • Reporter proteins include, but not limited to, fluorescent proteins (e.g. GFP, RFP, mCherry, YFP), > -galactosidase (D-gal), alkaline phosphatase (AP), chloramphenicol acetyl transferase (CAT), horseradish peroxidase (HRP).
  • affinity tags include polyhistidine (His tag), Glutathione S-Transferase (GST), Maltose Binding Protein (MBP), Calmodulin Binding Peptide (CBP), intein-chitin binding domain (intein-CBD), Streptavidin/B iotin-based tags, Epitope tags like FLAG, HA, c-myc, T7, Glu-Glu and many others.
  • Non-limiting examples of tags that enhance protein solubility include Streptococcal protein G (GB-1), Thioredoxin A, Glutathione S-Transferase (GST), Maltose Binding Protein (MBP), Small Ubiquitin-like Modifyer (SUMO), NusA , and any derivatives or variants thereof.
  • a fusion transposase as provided herein can comprise a TcBuster transposase sequence and a DNA sequence specific binding domain or a tag sequence fused together without any intermediate sequence (e.g., “back-to-back”).
  • a fusion transposase as provided herein can comprise a TcBuster transposase sequence and a DNA sequence specific binding domain or a tag sequence joined by a linker sequence.
  • a linker may serve primarily as a spacer between the first and second polypeptides.
  • a linker can be a short amino acid sequence to separate multiple domains in a single polypeptide.
  • a linker sequence can comprise linkers occurring in natural multi-domain proteins.
  • a linker sequence can comprise linkers artificially created. The choice of linker sequence may be based on the application of the fusion transposase.
  • a linker sequence can comprise 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.
  • the linker sequence may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 50 amino acids.
  • the linker sequence can comprise at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 15, at most 20, at most 30, at most 40, at most 50, or at most 100 amino acids.
  • a suitable linker sequence comprises GGGGS (SEQ ID NO: 11), one or more repeating GGGGS (SEQ ID NO: 11) units, such as (GGGGS)(SEQ ID NO: 11) repeating 2-8 times.
  • the linker comprises GGSGGSGGSGGSGTS (SEQ ID NO: 9), (GGGGGGGG) (SEQ ID NO: 12), GSAGSAAGSGEF (SEQ ID NO: 13), (GGGGS) 4 (SEQ ID NO: 14).
  • rigid linker sequences such as, but not limited EAAAK (SEQ ID NO: 15) or repeating sequences of EAAAK (SEQ ID NO: 15), including (EAAAK)(SEQ ID NO: 15) repeating 2-7 times.
  • EAAAK EAAAK
  • Pro-rich sequences like (XP)n with X designating any amino acid.
  • a TcBuster transposase sequence can be fused to the N-terminus of a DNA sequence specific binding domain or a tag sequence.
  • a TcBuster transposase sequence can be fused to the C-terminus of a DNA sequence specific binding domain or a tag sequence.
  • a third domain sequence or more of other sequences can be present in between the TcBuster transposase and the DNA sequence specific binding domain or the tag sequence, depending on the application of the fusion transposase.
  • polynucleotides e.g., nucleotide sequences, coding for a TcBuster transposase as provided herein.
  • the polynucleotides as provided herein comprise one or more codons that are favorable by a translation system of the organism whose cell the polynucleotide is delivered into.
  • a polynucleotide as provided herein can comprise one or more codons that are favorable by a human (e.g., Homo Sapiens)' translation system, when the polynucleotide is delivered to a human cell for genome editing purposes.
  • one or more codons in the polynucleotides coding for a TcBuster transposase as provided herein can be codons that are found at a higher frequency in the organism whose cell the polynucleotide is delivered into.
  • the TcBuster transposase as provided herein is delivered to a target cell in the form of a polynucleotide coding for it, and the codons of a high frequency in the target cell can be utilized by the translation system of the cell more efficiently as compared to the natural codons in the DNA coding for TcBuster transposase, thereby leading to an increased expression of the TcBuster transposase in the target cell.
  • the polynucleotides provided herein are codon optimized for expression in cells of a target species, e.g., human cells.
  • Another aspect of the present disclosure provides a cell expressing a vector comprising a polynucleotide disclosed herein.
  • a cell comprising a vector comprising a polynucleotide encoding a mutant TcBuster transposon provided herein.
  • the cell is a bacterial cell.
  • the bacterial cell is an E. coli strain.
  • the cell is from an E.
  • the cell is a eukaryotic cell.
  • the cell is a eukaryotic cell is selected from the group consisting of a CHO cell, a HEK293 cell, an Sf9 cell, a Saccharomyces cerevisiae cell, and a Pichia pastoris cell, and any derivatives or variants thereof.
  • TcBuster transposon that comprises a cassette cargo positioned between two inverted repeats.
  • a TcBuster transposon can be recognized by a TcBuster transposase as described herein, e.g., a TcBuster transposase can recognize the TcBuster transposon and catalyze transposition of the TcBuster transposon into a DNA sequence.
  • inverted repeats can refer to short sequence repeats flanking the transposase gene in a natural transposon or a cassette cargo in an artificially engineered transposon.
  • the two inverted repeats are generally required for the mobilization of the transposon in the presence of a corresponding transposase.
  • Inverted repeats as described herein may contain one or more direct repeat (DR) sequences. These sequences usually are embedded in the terminal inverted repeats (TIRs) of the elements.
  • DR direct repeat
  • TIRs terminal inverted repeats
  • carrier cassette as used herein can refer to a nucleotide sequence other than a native nucleotide sequence between the inverted repeats that contains the TcBuster transposase gene.
  • a cargo cassette can be artificially engineered.
  • a transposon described herein may contain a cargo cassette flanked by IR/DR sequences.
  • at least one of the repeats contains at least one direct repeat.
  • a transposon may contain a cargo cassette flanked by IRDR-L-Seql (SEQ ID NO: 3) and IRDR-R-Seql (SEQ ID NO: 4).
  • a left inverted repeat can comprise a sequence at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to IRDR-L-Seql (SEQ ID NO: 3).
  • a right inverted repeat can comprise a sequence at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to IRDR-R-Seql (SEQ ID NO: 4).
  • a right inverted repeat can comprise a sequence at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to IRDR-L-Seql (SEQ ID NO: 3).
  • a left inverted repeat can comprise a sequence at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to IRDR-R-Seql (SEQ ID NO: 4).
  • the terms “left” and “right”, as used herein, can refer to the 5 ’ and 3 ’ sides of the cargo cassette on the sense strand of the double strand transposon, respectively. It is also possible that a transposon may contain a cargo cassette flanked by IRDR-L-Seq2 (SEQ ID NO: 5) and IRDR-R-Seq2 (SEQ ID NO: 6).
  • a left inverted repeat can comprise a sequence at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to IRDR-L-Seq2 (SEQ ID NO: 5).
  • a right inverted repeat can comprise a sequence at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to IRDR-R-Seq2 (SEQ ID NO: 6).
  • a right inverted repeat can comprise a sequence at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to IRDR-L-Seq2 (SEQ ID NO: 5).
  • a left inverted repeat can comprise a sequence at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to IRDR-R-Seq2 (SEQ ID NO: 6).
  • a transposon can contain a cargo cassette flanked by IRDR-L-Seq3 (SEQ ID NO: 7) and IRDR-R- Seq3 (SEQ ID NO: 8).
  • a left inverted repeat can comprise a sequence at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to IRDR-L-Seq3 (SEQ ID NO: 7).
  • a right inverted repeat can comprise a sequence at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to 1RDR-R- Seq3 (SEQ ID NO: 8).
  • a right inverted repeat can comprise a sequence at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to IRDR-L-Seq3 (SEQ ID NO: 7).
  • a left inverted repeat can comprise a sequence at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to IRDR-R- Seq3 (SEQ ID NO: 8).
  • At least one of the two inverted repeats of a transposon described herein may contain a sequence that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 3-6.
  • At least one of inverted repeats of a transposon described herein may contain a sequence that is at least 80% identical to SEQ ID NO: 3 or 4. At least one of inverted repeats of a transposon described herein may contain a sequence that is at least 80% identical to SEQ ID NO: 5 or 6.
  • the choice of inverted repeat sequences may vary depending on the expected transposition efficiency, the type of cell to be modified, the transposase to use, and many other factors.
  • minimally sized transposon vector inverted terminal repeats that conserve genomic space may he used.
  • the ITRs of hAT family transposons diverge greatly with differences in right-hand and left-hand ITRs.
  • smaller ITRs consisting of just 100-200 nucleotides are as active as the longer native ITRs in hAT transposon vectors. These sequences may be consistently reduced while mediating hAT family transposition. These shorter ITRs can conserve genomic space within hAT transposon vectors.
  • the inverted repeats of a transposon provided herein can be about 50 to 2000 nucleotides, about 50 to 1000 nucleotides, about 50 to 800 nucleotides, about 50 to 600 nucleotides, about 50 to 500 nucleotides, about 50 to 400 nucleotides, about 50 to 350 nucleotides, about 50 to 300 nucleotides, about 50 to 250 nucleotides, about 50 to 200 nucleotides, about 50 to 180 nucleotides, about 50 to 160 nucleotides, about 50 to 140 nucleotides, about 50 to 120 nucleotides, about 50 to 110 nucleotides, about 50 to 100 nucleotides, about 50 to 90 nucleotides, about 50 to 80 nucleotides, about 50 to 70 nucleotides, about 50 to 60 nucleotides, about 75 to 750 nucleotides, about 75 to 450 nucleotides, about 75 to 325 nucleotides, about 75
  • a cargo cassette can comprise a promoter, a trans gene, or a combination thereof.
  • the expression of the transgene can be directed by the promoter.
  • a promoter can be any type of promoter available to one skilled in the art. Non-limiting examples of the promoters that can be used in a TcBuster transposon include EFS, CMV, MND, EFla, CAGGs, PGK, UBC, U6, Hl, MND, and Cumate.
  • the choice of a promoter to be used in a TcBuster transposition would depend on a number of factors, such as, but not limited to, the expression efficiency of the promoter, the type of cell to be genetically modified, and the desired transgene expression level.
  • a transgene in a TcBuster transposon can be any gene of interest and available to one skilled in the art.
  • a transgene can be derived from, or a variant of, a gene in nature, or can be artificially designed.
  • a transgene can be of the same species origin as the cell to be modified, or from different species.
  • a transgene can be a prokaryotic gene, or a eukaryotic gene.
  • a transgene can be a gene derived from a non-human animal, a plant, or a human being.
  • a transgene can comprise introns. Alternatively, a transgene may have introns removed or not present.
  • a transgene can code for a protein.
  • Exemplary proteins include, but are not limited to, a cellular receptor, an immunological checkpoint protein, a cytokine, or any combination thereof.
  • a cellular receptor as described herein can include, but not limited to a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof.
  • a cargo cassette as described herein may not contain a transgene coding for any type of protein product, but that is useful for other purposes.
  • a cargo cassette may be used for creating frameshift in the insertion site, for example, when it is inserted in an exon of a gene in the host genome. This may lead to a truncation of the gene product or a null mutation.
  • a cargo cassette may be used for replacing an endogenous genomic sequence with an exogenous nucleotide sequence, thereby modifying the host genome.
  • a transposon described herein may have a cargo cassette in either forward or reverse direction.
  • a cargo cassette has its own directionality.
  • a cargo cassette containing a transgene would have a 5’ to 3’ coding sequence.
  • a cargo cassette containing a promoter and a gene insertion would have promoter on the 5’ site of the gene insertion.
  • the term “forward direction”, as used herein, can refer to the situation where a cargo cassette maintains its directionality on the sense strand of the double strand transposon.
  • reverse direction as used herein, can refer to the situation where a cargo cassette maintains its directionality on the antisense strand of the double strand transposon.
  • a system can comprise a TcBuster transposase (e.g. a TcBuster transposase comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 1 and at least one N-terminal amino acid deletion) and a TcBuster transposon.
  • a system can be used to edit a genome of a host cell, disrupting or modifying an endogenous genomic region of the host cell, inserting an exogenous gene into the host genome, replacing an endogenous nucleotide sequence with an exogenous nucleotide sequence or any combination thereof.
  • a system for genome editing can comprise a mutant TcBuster transposase or fusion transposase as described herein, and a transposon recognizable by the mutant TcBuster transposase or the fusion transposase.
  • a mutant TcBuster transposase or the fusion transposase can be provided as a purified protein. Protein production and purification technologies are known to one skilled in the art. The purified protein can be kept in a different container than the transposon, or they can be kept in the same container.
  • a system for genome editing can comprise a polynucleotide encoding a mutant TcBuster transposase or fusion transposase as described herein, and a transposon recognizable by the mutant TcBuster transposase or the fusion transposase.
  • a polynucleotide of the system can comprise DNA that encodes the mutant TcBuster transposase or the fusion transposase.
  • a polynucleotide of the system can comprise messenger RNA (mRNA) that encodes the mutant TcBuster transposase or the fusion transposase.
  • mRNA messenger RNA
  • the mRNA can be produced by a number of approaches well known to one of ordinary skills in the art, such as, but not limited to, in vivo transcription and RNA purification, in vitro transcription, and de novo synthesis.
  • the mRNA can be chemically modified.
  • the chemically modified mRNA may be resistant to degradation than unmodified or natural mRNAs or may degrade more quickly.
  • the chemical modification of the mRNA may render the mRNA being translated with more efficiency.
  • Chemical modification of mRNAs can be performed with well-known technologies available to one skilled in the art, or by commercial vendors.
  • hAT transposase expression For many applications, safety dictates that the duration of hAT transposase expression be only long enough to mediate safe transposon delivery. Moreover, a pulse of hAT transposase expression that coincides with the height of transposon vector levels can achieve maximal gene delivery.
  • the implementations are made using available technologies for the in vitro transcription of RNA molecules from DNA plasmid templates.
  • the RNA molecules can be synthesized using a variety of methods for in vitro (e.g., cell free) transcription from a DNA copy. Methods to do this have been described and are commercially available. For example, the mMessage Machine in vitro transcription kit available through life technologies.
  • RNA molecules can be introduced into cells using any of many described methods for RNA transfection, which is usually non-toxic to most cells. Methods to do this have been described and are commercially available.
  • a transposon as described herein may be present in an expression vector.
  • the expression vector can be DNA plasmid.
  • the expression vector can be a mini-circle vector.
  • mini-circle vector can refer to small circular plasmid derivative that is free of most, if not all, prokaryotic vector parts (e.g., control sequences or non-functional sequences of prokaryotic origin). Under circumstances, the toxicity to the cells created by transfection or electroporation can be mitigated by using the “mini-circles” as described herein.
  • a mini-circle vector can be prepared by well-known molecular cloning technologies available. First, a 'parental plasmid' (bacterial plasmid with insertion, such as transposon construct) in bacterial, such as E. coli, can be produced, which can be followed by induction of a site-specific recombinase. These steps can then be followed by the excision of prokaryotic vector parts via two recombinase-target sequences at both ends of the insert, as well as recovery of the resulting mini-circle vector. The purified mini-circle can be transferred into the recipient cell by transfection or lipofection and into a differentiated tissue by, for instance, jet injection.
  • a mini-circle containing TcBuster transposon can have a size about 1.5kb, about 2 kb, about 2.2 kb, about 2.4 kb, about 2.6 kb, about 2.8 kb, about 3 kb, about 3.2 kb, about 3.4 kb, about 3.6 kb, about 3.8 kb, about 4 kb, about 4.2 kb, about 4.4 kb, about 4.6 kb, about 4.8 kb, about 5 kb, about 5.2 kb, about 5.4 kb, about 5.6 kb, about 5.8 kb, about 6 kb, about 6.5 kb, about 7 kb, about 8 kb, about 9 kb, about 10 kb, about 12 kb, about 25 kb, about 50 kb, or a value between any two of these numbers.
  • a mini-circle containing TcBuster transposon as provided herein can have a size at most 2.1 kb, at most 3.1 kb, at most 4.1 kb, at most 4.5 kb, at most 5.1 kb, at most 5.5 kb, at most 6.5 kb, at most 7.5 kb, at most 8.5 kb, at most 9.5 kb, at most 11 kb, at most 13 kb, at most 15 kb, at most 30 kb, or at most 60 kb.
  • a system as described herein may contain a polynucleotide encoding a mutant TcBuster transposase or fusion transposase as described herein, and a transposon, which are present in a same expression vector, e.g. plasmid.
  • a method of genetic engineering can comprise introducing into a cell a TcBuster transposase and a transposon recognizable by the TcBuster transposase.
  • a method of genetic engineering can also be performed in a cell-free environment.
  • a method of genetic engineering in a cell-free environment can comprise combining a TcBuster transposase, a transposon recognizable by the transposase, and a target nucleic acid into a container, such as a well or tube.
  • a method described herein can comprises introducing into a cell a mutant TcBuster transposase provided herein and a transposon recognizable by the mutant TcBuster transposase.
  • a method of genome editing can comprise: introducing into a cell a fusion transposase provided herein and a transposon recognizable by the fusion transposase.
  • the mutant TcBuster transposase or the fusion transposase can be introduced into the cell either as a protein or via a polynucleotide that encodes for the mutant TcBuster transposase or the fusion transposase.
  • the polynucleotide as discussed above, can comprise a DNA or an mRNA that encodes the mutant TcBuster transposase or the fusion transposase.
  • the mutant TcBuster transposase or the fusion transposase is introduced into the cell as RNA (e.g. mRNA).
  • the mRNA comprises a 5’ cap.
  • RNA cap or a 5’ cap refers to an altered nucleotide on the 5’ end of an RNA.
  • the 5’ cap comprises a 7’ methylguanylate cap (m7G, cap-0).
  • the 5’ cap comprises a methylation of the 2’ hydroxy-groups of the first ribose sugar (cap-1) or the first two ribose sugars (cap-2) on the 5’ end of mRNA.
  • the 5’ cap comprises a 5 ’-trimethylguanosine cap, a 5 ’-monomethylphosphate cap, a NAD+ cap, a NADH cap, or a 3’ dephospho-coenzyme A cap.
  • the 5’ cap protects the mRNA from degradation during transcription.
  • the 5’ cap provides resistance to exonucleases.
  • the TcBuster transposase or the fusion transposase can be transfected into a host cell as a protein, and the concentration of the protein can be at least 0.05nM, at least 0.1 nM, at least 0.2 nM, at least 0.5 nM, at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 DM, at least 2
  • ⁇ M at least 5 DM, at least 7.5 DM, at least 10 DM, at least 15 DM, at least 20 DM, at least 25
  • the concentration of the protein can be around 1 DM to around 50 UM, around 2 ⁇ M to around 25 DM, around 5 DM to around 12.5 DM, or around 7.5 DM to around 10 DM.
  • the TcBuster transposase or the fusion transposase can be transfected into a host cell through a polynucleotide, and the concentration of the polynucleotide can be at least about 5 ng/ml, 10 ng/ml, 20 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 80 ng/ml, 100 ng/ml, 120 ng/ml, 150 ng/ml, 180 ng/ml, 200 ng/ml, 220 ng/ml, 250 ng/ml, 280 ng/ml, 300 ng/ml, 500 ng/ml, 750 ng/ml, 1 pg /ml, 2 pg /ml, 3 pg /ml, 5 pg/ml, 50 pg/ml, 100 pg/ml, 150 pg/ml, 200
  • the concentration of the polynucleotide can be between about 5-25 pg/ml, 25-50 pg/ml, 50-100 pg/ml, 100-150 pg/ml, 150-200 pg/ml, 200-250 pg/ml, 250-500 pg/ml, 5-800 pg/ml, 200-800 pg/ml, 250-800 pg/ml, 400-800 pg/ml, 500-800 pg/ml, or any range derivable therein.
  • the transposon is present in a separate expression vector than the transposase, and the concentration of the transposon can be at least about 5 ng/ml, 10 ng/ml, 20 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 80 ng/ml, 100 ng/ml, 120 ng/ml, 150 ng/ml, 180 ng/ml, 200 ng/ml, 220 ng/ml, 250 ng/ml, 280 ng/ml, 300 ng/ml, 500 ng/ml, 750 ng/ml, 1 pg /ml, 2 pg /ml, 3 pg /ml, 5 pg/ml, 50 pg/ml, 100 pg/ml, 150 pg/ml, 200 pg/ml, 250 pg/ml, 300 pg/ml, 350 pg/m
  • the concentration of the transposon can be between about 5-25 pg/ml, 25-50 pg/ml, 50-100 pg/ml, 100-150 pg/ml, 150-200 pg/ml, 200-250 pg/ml, 250-500 pg/ml, 5-800 pg/ml, 200-800 pg/ml, 250-800 pg/ml, 400-800 pg/ml, 500-800 pg/ml, or any range derivable therein.
  • the ratio of the transposon versus the polynucleotide coding for the transposase is at most 10000, at most 5000, at most 1000, at most 500, at most 200, at most 100, at most 50, at most 20, at most 10, at most 5, at most 2, at most 1, at most 0.1, at most 0.05, at most 0.01, at most 0.001, at most 0.0001, or any number in between any two thereof.
  • the transposon and the polynucleotide coding for the transposase are present in the same expression vector, and the concentration of the expression vector containing both transposon and the polynucleotide encoding transposase can be at least about 5 ng/ml, 10 ng/ml, 20 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 80 ng/ml, 100 ng/ml, 120 ng/ml, 150 ng/ml, 180 ng/ml, 200 ng/ml, 220 ng/ml, 250 ng/ml, 280 ng/ml, 300 ng/ml, 500 ng/ml, 750 ng/ml, 1 pg /ml, 2 pg /ml, 3 pg /ml, 5 pg/ml, 50 pg/ml, 100 pg/ml, 150
  • the concentration of the expression vector containing both transposon and the polynucleotide encoding transposase can be between about 5-25 pg/ml, 25-50 pg/ml, 50-100 pg/ml, 100-150 pg/ml, 150-200 pg/ml, 200-250 pg/ml, 250-500 pg/ml, 5-800 pg/ml, 200-800 pg/ml, 250-800 pg/ml, 400-800 pg/ml, 500-800 pg/ml, or any range derivable therein.
  • the amount of polynucleic acids that may be introduced into the cell by electroporation may be varied to optimize transfection efficiency and/or cell viability. In some cases, less than about 100 pg of nucleic acid may be added to each cell sample (e.g., one or more cells being electroporated).
  • dsDNA 1 microgram of dsDNA may be added to each cell sample for electroporation.
  • the amount of polynucleic acids (e.g., dsDNA) required for optimal transfection efficiency and/or cell viability may be specific to the cell type.
  • the subject matter disclosed herein may find use in genome editing of a wide range of various types of host cells.
  • the host cells may be from eukaryotic organisms.
  • the cells may be from a mammal origin.
  • the cells may be from a human origin.
  • cell line and “immortalized cell line”, as used herein interchangeably, can refer to a population of cells from an organism which would normally not proliferate indefinitely but, due to mutation, may have evaded normal cellular senescence and instead can keep undergoing division.
  • the subject matter provided herein may find use in a range of common established cell lines, including, but not limited to, human BC-1 cells, human BIAB cells, human IM-9 cells, human liyoye cells, human K-562 cells, human LCL cells, mouse MPC-11 cells, human Raji cells, human Ramos cells, mouse Ramos cells, human RPMI8226 cells, human RS4- 11 cells, human SKW6.4 cells, human Dendritic cells, mouse P815 cells, mouse RBL-2H3 cells, human HL-60 cells, human NAMALWA cells, human Macrophage cells, mouse RAW 264.7 cells, human KG-1 cells, mouse Ml cells, human PBMC cells, mouse BW5147 (T200-A)5.2 cells, human CCRF-CEM cells, mouse EL4 cells, human lurkat cells, human SCID.adh cells, human U-937 cells or any combination of cells thereof.
  • primary cells can refer to cells taken directly from an organism, typically living tissue of a multicellular organism, such as animals or plants.
  • primary cells may be established for growth in vitro.
  • primary cells may be just removed from the organism and have not been established for growth in vitro yet before the transfection.
  • the primary cells can also be expanded in vitro, i.e. primary cells may also include progeny cells that are generated from proliferation of the cells taken directly from an organism.
  • the progeny cells do not exhibit the indefinite proliferative property as cells in established cell lines.
  • the host cells may be human primary T cells, while prior to the transfection, the T cells have been exposed to stimulatory factor(s) that may result in T cell proliferation and expansion of the cell population.
  • the cells to be genetically modified may be primary cells from tissues or organs, such as, hut not limited to, brain, lung, liver, heart, spleen, pancreas, small intestine, large intestine, skeletal muscle, smooth muscle, skin, bones, adipose tissues, hairs, thyroid, trachea, gall bladder, kidney, ureter, bladder, aorta, vein, esophagus, diaphragm, stomach, rectum, adrenal glands, bronchi, ears, eyes, retina, genitals, hypothalamus, larynx, nose, tongue, spinal cord, or ureters, uterus, ovary, testis, and any combination thereof.
  • tissues or organs such as, hut not limited to, brain, lung, liver, heart, spleen, pancreas, small intestine, large intestine, skeletal muscle, smooth muscle, skin, bones, adipose tissues, hairs, thyroid, trachea, gall bladder, kidney
  • the cells may include, but not limited to, hematocyte, trichocyte, keratinocyte, gonadotrope, corticotrope, thyrotrope, somatotrope, lactotroph, chromaffin cell, parafollicular cell, glomus cell, melanocyte, nevus cell, merkel cell, odontoblast, cementoblast, corneal keratocyte, retina muller cell, retinal pigment epithelium cell, neuron, glia, ependymocyte, pinealocyte, pneumocyte, clara cell, goblet cell, G cell, D cell, Enterochromaffin-like cell, gastric chief cell, parietal cell, foveolar cell, K cell, D cell, I cell, paneth cell, enterocyte, microfold cell, hepatocyte, hepatic stellate cell, cholecystocyte, centroacinar cell, pancreatic stellate cell, pancreatic a cell, pancreatic P
  • the cell to be modified may be a stem cell, such as, but not limited to, embryonic stem cell, hematopoietic stem cell, epidermal stem cell, epithelial stem cell, bronchoalveolar stem cell, mammary stem cell, mesenchymal stem cell, intestine stem cell, endothelial stem cell, neural stem cell, olfactory adult stem cell, neural crest stem cell, testicular cell, and any combination thereof.
  • the cell can be an induced pluripotent stem cell that is derived from any type of tissue.
  • the cell to be genetically modified may be a mammalian cell.
  • the cell may be an immune cell.
  • Non-limiting examples of the cell can include a B cell, a basophil, a dendritic cell, an eosinophil, a gamma delta T cell, a granulocyte, a helper T cell, a Langerhans cell, a lymphoid cell, an innate lymphoid cell (ILC), a macrophage, a mast cell, a megakaryocyte, a memory T cell, a monocyte, a myeloid cell, a natural killer T cell, a neutrophil, a precursor cell, a plasma cell, a progenitor cell, a regulatory T-cell, a T cell, a thymocyte, any differentiated or de-differentiated cell thereof, or any mixture or combination of cells thereof.
  • the cell may be a T cell. In some embodiments, the cell may be a primary T cell. In certain cases, the cell may be an antigen-presenting cell (APC). In some embodiments, the cell may be a primary APC.
  • the APCs in connection with the present disclosure may be a dendritic cell, macrophage, B cell, other non-professional APCs, or any combination thereof.
  • the cell may be an ILC (innate lymphoid cell), and the ILC can be a group 1 ILC, a group 2 ILC, or a group 3 ILC.
  • Group 1 ILCs may generally be described as cells controlled by the T-bet transcription factor, secreting type-1 cytokines such as IFN-gamma and TNF-alpha in response to intracellular pathogens.
  • Group 2 ILCs may generally be described as cells relying on the GATA-3 and ROR-alpha transcription factors, producing type-2 cytokines in response to extracellular parasite infections.
  • Group 3 ILCs may generally be described as cells controlled by the ROR-gamma t transcription factor, and produce IL- 17 and/or IL-22.
  • the cell may be a cell that is positive or negative for a given factor.
  • a cell may be a CD3+ cell, CD3- cell, a CD5+ cell, CD5- cell, a CD7+ cell, CD7- cell, a CD14+ cell, CD14- cell, CD8+ cell, a CD8- cell, a CD103+ cell, CD103- cell, CD1 lb+ cell, CD1 lb- cell, a BDCA1+ cell, a BDCA1- cell, an L-selectin+ cell, an L-selectin- cell, a CD25+, a CD25- cell, a CD27+, a CD27- cell, a CD28+ cell, CD28- cell, a CD44+ cell, a CD44- cell, a CD44- cell, a CD56+ cell, a CD56- cell, a CD57+ cell, a CD57- cell, a CD62L+ cell, a CD62
  • the cell may be positive or negative for any factor known in the art.
  • the cell may be positive for two or more factors.
  • the cell may be CD4+ and CD8+.
  • the cell may be negative for two or more factors.
  • the cell may be CD25-, CD44-, and CD69-.
  • the cell may be positive for one or more factors, and negative for one or more factors.
  • a cell may be CD4+ and CD8-.
  • cells used in any of the methods disclosed herein may be a mixture (e.g., two or more different cells) of any of the cells disclosed herein.
  • a method of the present disclosure may comprise cells, and the cells are a mixture of CD4+ cells and CD8+ cells.
  • a method of the present disclosure may comprise cells, and the cells are a mixture of CD4+ cells and naive cells.
  • the transposase and the transposon can be introduced into a cell through a number of approaches.
  • the term “transfection” and its grammatical equivalents as used herein can generally refer to a process whereby nucleic acids are introduced into eukaryotic cells.
  • transfection methods that can be used in connection with the subject matter can include, but not limited to, electroporation, microinjection, calcium phosphate precipitation, cationic polymers, dendrimers, liposome, microprojectile bombardment, fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, nucleofection, or any combination thereof.
  • the transposase and transposon described herein can be transfected into a host cell through electroporation.
  • transfection can also be done through a variant of electroporation method, such as nucleofection (also known as NucleofectorTM technology).
  • electroporation and its grammatical equivalents as used herein can refer to a process whereby an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or DNA to be introduced into the cell.
  • the electric filed is often provided in the form of “pulses” of very brief time periods, e.g. 5 milliseconds, 10 milliseconds, and 50 milliseconds.
  • electroporation temporarily opens up pores in a cell's outer membrane by use of pulsed rotating electric fields.
  • Methods and apparatus used for electroporation in vitro and in vivo are also well known.
  • Various electric parameters can be selected dependent on the cell type being electroporated and physical characteristics of the molecules that are to be taken up by the cell, such as pulse intensity, pulse length, number of pulses).
  • compositions e.g., mutant TcBuster transposases, fusion transposases, TcBuster transposons
  • systems and methods provided herein may find use in a wide range of applications relating to genome editing, in various aspects of modem life.
  • advantages of the subject matter described herein may include, but not limited to, reduced costs, regulatory consideration, lower immunogenicity and less complexity.
  • a significant advantage of the present disclosure is the high transposition efficiency.
  • Another advantage of the present disclosure in many cases, is that the transposition system provided herein can be “tunable”, e.g., transposition can be designed to target select genomic region rather than random insertion.
  • One particular example includes generation of genetically modified primary leukocytes using the methods provided herein, and administering the genetically modified primary leukocytes to a patient in need thereof.
  • the generation of genetically modified primary leukocytes can include introducing into a leukocyte a transposon and a mutant TcBuster transposase or the fusion transposase as described herein, which can recognize the transposon, thereby generating a genetically modified leukocyte.
  • the transposon may comprise a transgene.
  • the transgene can be a cellular receptor, an immunological checkpoint protein, a cytokine, and any combination thereof.
  • Another non-limiting example is related to create genetically modified organisms for agriculture, food production, medicine, and pharmaceutics.
  • the species that can be genetically modified span a wide range, including, but not limited to, plants and animals.
  • the genetically modified organisms such as genetically modified crops or livestock, may be modified in a certain aspect of their physiological properties.
  • food crops include resistance to certain pests, diseases, or environmental conditions, reduction of spoilage, or resistance to chemical treatments (e.g. resistance to a herbicide), or improving the nutrient profile of the crop.
  • non-food crops include production of pharmaceutical agents, biofuels, and other industrially useful goods, as well as for bioremediation.
  • Examples in livestock include resistance to certain parasites, production of certain nutrition elements, increase in growth rate, and increase in milk production.
  • the term “about” and its grammatical equivalents in relation to a reference numerical value and its grammatical equivalents as used herein can include a range of values plus or minus 10% from that value.
  • the amount “about 10” includes amounts from 9 to 11.
  • the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.
  • TcBuster and other transposases are not soluble enough for scalable purification.
  • This example addresses this issue by identifying a region of TcBuster that can be removed to yield a more soluble protein when expressed in cells. Specifically, Alphafold, multiple sequence alignments, and NLS predictive software was used to identify a region of TcBuster that can be removed. Deleting amino acids 2-42 and 2-48 of TcBuster yielded significantly more soluble expression in E. coli.
  • TcBuster truncation in which N- terminal amino acids 2-42 were truncated
  • TcBuster truncation in which N-terminal amino acids 2-48 were truncated.
  • the truncated TcBuster proteins were expressed in E. coli and solubility was assessed by western blot. As shown in FIG. 6, the truncated versions of TcBuster were shown to be more soluble in E. Coli than full length TcBuster. As shown in FIG. 7, the increased solubility seen in the truncated proteins was consistent across a variety of E. Coli strains. As shown in FIG. 8, this increased solubility was also shown to be consistent across multiple experiments.
  • the activity of truncated TcBuster was also compared to activity of full length protein. As shown in FIG. 9A-8B, the truncated TcBuster proteins were shown to be as active as full length protein. As shown in FIG. 9C, the truncated TcBuster provided higher expression populations of cells earlier on in the experiment compared to full length. [00146] Removing cystines from TcBuster did not result in improved solubility compared to wildtype. Moreover, use of FireProt (PMID: 28449074) to predict stabilizing mutations did not increase solubility.
  • FireProt PMID: 28449074

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Abstract

L'invention concerne des transposases TcBuster mutantes présentant une solubilité améliorée. Selon certains aspects, l'invention concerne des transposases TcBuster mutantes comprenant une séquence d'acides aminés qui est identique à au moins 70 % à SEQ ID NO : 1 ; et au moins une délétion d'acide aminé N-terminal. Les transposases TcBuster mutantes fournies ici sont solubles dans une cellule hôte telle que E. Coli.
PCT/US2024/045520 2023-09-07 2024-09-06 Transposase tcbuster mutante à solubilité améliorée Pending WO2025054409A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180216087A1 (en) * 2016-12-16 2018-08-02 B-Mogen Biotechnologies, Inc. Enhanced hAT Family Transposon-Mediated Gene Transfer and Associated Compositions, Systems and Methods
WO2019246486A2 (fr) * 2018-06-21 2019-12-26 B-Mogen Biotechnologies, Inc. Transfert amélioré de gènes médié par transposon de la famille hat et compositions, systèmes et méthodes associés
WO2020077360A1 (fr) * 2018-10-12 2020-04-16 Poseida Therapeutics, Inc. Compositions de cellules souches hématopoïétiques, leurs procédés de production et d'utilisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180216087A1 (en) * 2016-12-16 2018-08-02 B-Mogen Biotechnologies, Inc. Enhanced hAT Family Transposon-Mediated Gene Transfer and Associated Compositions, Systems and Methods
WO2019246486A2 (fr) * 2018-06-21 2019-12-26 B-Mogen Biotechnologies, Inc. Transfert amélioré de gènes médié par transposon de la famille hat et compositions, systèmes et méthodes associés
WO2020077360A1 (fr) * 2018-10-12 2020-04-16 Poseida Therapeutics, Inc. Compositions de cellules souches hématopoïétiques, leurs procédés de production et d'utilisation

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