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WO2014121222A1 - Endonucléase pour édition génomique - Google Patents

Endonucléase pour édition génomique Download PDF

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Publication number
WO2014121222A1
WO2014121222A1 PCT/US2014/014491 US2014014491W WO2014121222A1 WO 2014121222 A1 WO2014121222 A1 WO 2014121222A1 US 2014014491 W US2014014491 W US 2014014491W WO 2014121222 A1 WO2014121222 A1 WO 2014121222A1
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Prior art keywords
dna
gene
domain
nucleic acid
cell
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English (en)
Inventor
David R. EDGELL
Benjamin P. KLEINSTIVER
Li Wang
Adam J. Bogdanove
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University of Western Ontario
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University of Western Ontario
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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
    • 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
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1082—Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors

Definitions

  • the present application relates generally to endonucleases useful for gene editing.
  • the other architecture utilizes the reprogrammable DNA-binding specificity of zinc-finger proteins or the DNA-binding domains of transcription activator-like effectors (TAL-effectors) that are fused to the non-specific nuclease domain of the type IIS restriction enzyme Fokl to create chimeric zinc-finger nucleases (ZFNs) or TAL-effector nucleases (TALENs).
  • TAL-effectors transcription activator-like effectors
  • Fokl transcription activator-like effectors
  • ZFNs chimeric zinc-finger nucleases
  • TALENs TAL-effector nucleases
  • GIY-YIG nuclease domain is associated with a variety of proteins with diverse cellular functions.
  • the small (-100 aa) globular GIY-YIG domain is characterized by a structurally conserved central three-stranded antiparallel ⁇ sheet, with catalytic residues positioned to utilize a single metal ion to promote DNA hydrolysis.
  • the GIY-YIG homing endonucleases typified by the isoschizomers I-TevI (a double-strand DNA endonuclease encoded by the mobile td intron of phage T4), I-Bmol and I-Tulal bind DNA as monomers. It is unknown, however, if GIY-YIG homing endonucleases function as monomers in all steps of the reaction, as it is possible that dimerization between GIY-YIG nuclease domains is necessary for efficient DNA hydrolysis, as is the case with Fokl. Notably, GIY-YIG homing endonucleases require a specific DNA sequence to generate a DSB.
  • the bottom ( ⁇ ) and top (j) strand nicking sites lie within a 5'-CN ⁇ N
  • CNNNG 5'-CN ⁇ N
  • the present invention provides chimeric endonucleases and methods of making and using such chimeric endonucleases.
  • the present invention provides a chimeric endonuclease comprising at least a nuclease domain and a DNA-targeting domain.
  • the nuclease domain has the ability to cleave double-stranded DNA, typically at a specific DNA sequence.
  • the nuclease is capable of cleaving double-stranded DNA as a monomer.
  • the nuclease domain may be derived from a homing endonuclease.
  • Suitable examples of homing endonucleases include, but are not limited to, homing endonucleases of the LAGLIDADG, HNH, His-Cys box, and GIY-YIG families.
  • a chimeric endonuclease of the invention comprises a nuclease domain derived from a homing endonulcease of the GIY-YIG family.
  • Suitable examples of homing endonucleases of the GIY-YIG family include, but are not limited to, I-TevI and I-Bmol.
  • a chimeric endonuclease of the invention comprises the nuclease domain of I-TevI.
  • Chimeric endonucleases of the invention may be provided as part of a composition, for example, a pharmaceutical composition.
  • the present invention also provides cells, cell lines and transgenic organisms (e.g., plants, fungi, animals) comprising one or more chimeric endonucleases of the invention.
  • Suitable cells include, but are not limited to, mammalian cells (e.g., mouse cells, human cells, rat cells, etc.) which may be stem cells, avian cells, plant cells, bacterial cell, fungal cells (e.g., yeast cells), and any other type of cell known to those skilled in the art.
  • DNA-binding domains any specific DNA-binding domain known to those skilled in the art may be used as a DNA-targeting domain in the practice of the present invention.
  • TAL domains such as PthXo l and AvrBs3 (from Xanthamonas campestris); zinc finger domains, e.g. ryA zinc finger binding domain and ryB zinc finger binding domain, and other distinct DNA-binding domains, such as the binding domain in LAGLIDADG homing endonucleases, for example I-Onul.
  • the entire LAGLIDADG homing endonuclease, not just the binding domain, may be used as a DNA-targeting domain in the practice of the present invention.
  • the nuclease activity of the LAGLIDADG endonuclease may be disrupted, for example, with a point mutation, such that it acts as a DNA-binding platform only.
  • a chimeric endonuclease of the invention may comprise one or more additional domains.
  • additional domains include, but are not limited to, linking domains and functional domains.
  • linking domains may be disposed between two functional domains, for example, between a nuclease domain and a DNA-targeting domain.
  • Other functional domains include domains comprising nuclear localization signals, transcription activating domains, dimerization domains, and other functional domains known to those skilled in the art.
  • the present invention also provides nucleic acid molecules encoding the chimeric endonucleases of the invention. Such molecules may be DNA or RNA.
  • DNA molecules will comprise one or more promoter regions operably linked to a nucleic acid sequence encoding all or a portion of a chimeric endonuciease of the invention.
  • Nucleic acid molecules of the invention may be provided as part of a larger nucleic acid molecule, for example, an expression vector. Suitable expression vectors include, but are not limited to, plasmid vectors, viral vectors, and retroviral vectors. Nucleic acid molecules of the invention may be provided as part of a composition, for example, a pharmaceutical composition.
  • the present invention also provides cells, cell lines and transgenic organisms (e.g., plants, fungi, animals) comprising one or more nucleic acid molecules of the invention.
  • Suitable cells include, but are not limited to, mammalian cells (e.g., mouse cells, human cells, rat cells, etc.) which may be stem cells, avian cells, plant cells, insect cells, bacterial cells, fungal cells (e.g., yeast cells), and any other type of cell known to those skilled in the art.
  • mammalian cells e.g., mouse cells, human cells, rat cells, etc.
  • stem cells e.g., avian cells, plant cells, insect cells, bacterial cells, fungal cells (e.g., yeast cells), and any other type of cell known to those skilled in the art.
  • a method of cleaving a target nucleic acid comprising the step of exposing target nucleic acid to a chimeric endonuciease as defined above, wherein the DNA targeting domain of the endonuciease binds to the target nucleic acid and the nuclease domain cleaves the target nucleic acid.
  • the target nucleic acid may be a gene of interest in a cell.
  • methods of the invention may be used in genomic editing applications.
  • a method of this type will comprise introducing, into the cell, one or more one chimeric endonucleases of the invention that bind to a target nucleic acid sequence in the gene (or nucleic acid molecules encoding such chimeric endonucleases under conditions resulting in expression of the chimeric endonucleases), wherein the DNA-targeting domain of the endonuciease binds to the target nucleic acid sequence and the nuclease domain cleaves the target nucleic acid.
  • cleavage of the gene results in disrupting the function of the gene as repair of the double-stranded break introduced by the chimeric endonuciease of the invention may result in one or more insertions and or deletions of nucleotides at the site of the break.
  • the present invention provides a method for introducing an exogenous nucleotide sequence into the genome of a cell.
  • Such methods typically comprise, introducing, into the cell, one or more chimeric endonucleases of the invention (or nucleic acid molecules encoding such chimeric endonucleases under conditions resulting in expression of the chimeric endonucleases), wherein the DNA-targeting domain of the endonuclease binds to the target nucleic acid and the nuclease domain cleaves the target nucleic acid, and contacting the cell with an exogenous polynucleotide; under conditions such that the exogenous polynucleotide is integrated into the genome by homologous recombination.
  • the exogenous polynucleotide may comprise a nucleic acid sequence that is capable of interacting with a protein.
  • Suitable examples of such sequences include, but are not limited to, recognition sites (e.g., endonuclease recognition sites, recombinase recognition sites), promoter sequences, and protein binding sites.
  • the present invention provides a chimeric endonuclease.
  • a chimeric endonuclease typically comprises a nuclease domain and a DNA-targeting domain.
  • the chimeric endonuclease is capable of cleaving double-stranded DNA as a monomer.
  • the nuclease domain is a site-specific nuclease domain, which may be from a homing endonuclease.
  • a suitable example of a homing endonuclease is a GIY-YIG homing endonuclease, for example 1-TevI.
  • a chimeric endonuclease of the invention may further comprise a linking domain.
  • the DNA-targeting domain is a TAL domain.
  • the chimeric endonuclease comprises a I-Tevl nuclease domain and a TAL DNA-targeting domain.
  • I-Tevl nuclease is N-terminal to the TAL domain.
  • the present invention also provides nucleic acid molecules encoding chimeric endonucleases as described above.
  • the present invention provides a method of inactivating a gene.
  • Such methods typically comprise introducing into a cell comprising the gene a nucleic acid molecule encoding a chimeric endonuclease as described above under conditions causing the expression of the chimeric endonuclease.
  • the chimeric endonuclease comprises a DNA-targeting domain that binds the gene and cleaves it.
  • the expression of the chimeric endonuclease is transient, in some embodiments, the cell is a plant cell.
  • the nucleic acid molecule is an mRNA.
  • the present invention provides a method of altering a gene in a cell.
  • Such methods typically comprise introducing a first nucleic acid molecule encoding a chimeric endonuclease as described above into a cell comprising the gene under conditions causing the expression of the chimeric endonuclease and cleavage of the gene.
  • Such methods may further comprise introducing a second nucleic acid molecule into the cell.
  • the second nucleic acid molecule comprises a region having a nucleotide sequence that has a high degree of sequence identity to all or a portion of the gene in the region of the cleavage site.
  • the second nucleic acid molecule is introduced under conditions causing homologous recombination to occur between the second nucleic acid molecule and the gene.
  • the region of high sequence identity comprises a sequence that is highly identical to all or a portion of the sequence of the gene.
  • the region of high sequence identity of the second nucleic acid molecule is not 100% identical to the corresponding region of the gene. Instead the region comprises an altered sequence when compared to the gene of interest.
  • the region may comprise one or more mutations that will result in changes to one or more amino acids in a protein encoded by the gene.
  • the chimeric endonuclease is transiently expressed in the cell.
  • the first nucleic acid molecule is mRNA.
  • the second nucleic acid molecule is a linear DNA molecule.
  • the cell is a plant cell.
  • the present invention provides a method for deleting all or a portion of a gene in a cell.
  • Such methods typically comprise introducing a first nucleic acid molecule encoding a chimeric endonuclease as described above into a cell comprising the gene under conditions causing expression of the chimeric endonuclease and cleavage of the gene.
  • a second nucleic acid molecule comprising a region having a nucleotide sequence that has a high degree of sequence identity to the gene in the region of the cleavage site is introduced into the cell under conditions causing homologous recombination to occur between the second nucleic acid molecule and the gene.
  • the region of high sequence identity lacks the sequence of the gene adjacent to the cleavage site.
  • the region of high sequence identity comprises a sequence that is highly identical to all or a portion of the sequence of the gene.
  • the region of high sequence identity of the second nucleic acid molecule is not 100% identical to the corresponding region of the gene. Instead the region comprises an altered sequence when compared to the gene of interest.
  • the region comprises one or more mutations that will result in changes to one or more amino acids in a protein encoded by the gene.
  • the chimeric endonuclease is transiently expressed in the cell.
  • the first nucleic acid molecule is mRNA.
  • the second nucleic acid molecule is a linear DNA molecule.
  • the cell is a plant cell.
  • the present invention provides a method for making a cell having an altered genome.
  • Such methods typically comprise introducing into the cell a first nucleic acid molecule encoding a chimeric endonuclease as described above under conditions causing expression of the chimeric endonuclease and cleavage of the gene.
  • the altered genome comprises an inactivated gene.
  • Methods of making a cell having an altered genome may also comprise introducing into the cell a second nucleic acid molecule comprising a region having a nucleotide sequence that has a high degree of sequence identity to the gene in the region of the cleavage site.
  • the second nucleic acid molecule is introduced into the cell under conditions causing homologous recombination between the gene and the second nucleic acid, wherein the region of high sequence identity comprises an altered sequence when compared to the gene.
  • the region of high sequence identity comprises a sequence that is highly identical to all or a portion of the sequence of the gene.
  • the region comprises one or more mutations that will result in changes to one or more amino acids in a protein encoded by the gene.
  • the nucleotide sequence of the region lacks the sequence of the gene adjacent to the cleavage site.
  • the chimeric endonuclease is transiently expressed in the cell.
  • the first nucleic acid molecule is mRNA.
  • the second nucleic acid molecule is a linear DNA molecule.
  • the cell is a plant cell.
  • the present invention provides a nucleic acid substrate for the chimeric endonuclease as described above. Such a substrate will typically comprise a cleavage motif of the nuclease domain, a spacer that correlates with the linking domain and a binding site for the DNA-targeting domain.
  • the present invention also provides cells, for example plant cells, incorporating the substrate.
  • kits comprising nucleic acid molecules encoding the chimeric endonucleases described above and a substrate for the chimeric endonuclease.
  • the invention provides kits comprising the chimeric endonucleases of the invention. Kits of the invention can be used for genomic editing using the methods described above.
  • Figure 1 illustrates that I-Bmol functions as a monomer.
  • Figure 1A provides graphs of progress curves of initial reaction velocity for eight I-Bmol concentrations with fixed amount ( ⁇ ) of pBmolHS target site plasmid (left) and plot of initial velocity versus I-Bmol protein concentration (right).
  • Figure IB provides graphs showing results of time course assays showing cleavage of 1 - or 2- site target plasmids by I-Bmol;
  • Figure 2 schematically illustrates the design and functionality of chimeric GIY-YIG endonucleases of the invention.
  • Figure 2a provides a schematic modeling of a Tev-zinc finger fusion with DNA substrate using structures of the I- TevI catalytic domain (PDB 1MK0), the I-Tevl DNA-binding domain co-crystal (PDB 113J), and the Zif268 co-crystal (PDB 1 AAY).
  • Figure 2b (upper) provides a schematic of a chimeric I-Tevl endonuclease-ryA construct showing the fusion point as the last I-Tevl amino acid, with an optional 2xGlycine or 4xGlycine linker and 6xHis tag at the C-terminal end, and (lower) a Tev-ryA substrate including 33-nts of the top strand of the I-Tevl td homing site substrate (T21.33), fused to the 5' end of the ryA-binding site.
  • the substrate is numbered from the first base of the td homing site sequence (note that is numbering scheme is reverse of that used for the native td homing site).
  • Figure 2c (upper) provides a schematic of a chimeric I-Bmol endonuclease-ryA construct showing the fusion point as the last I- Bmol amino acid, with an optional 2xGlycine or 4xGlycine linker and 6xHis tag at the C-terminal end, and (lower) a I-BmoI-ryA substrate including 33-nts of the top strand of the I-Bmol homing site substrate (BZ1.33), fused to the 5' end of the ryA- binding site.
  • Figure 2d provides a schematic representation of the two plasmids used in the genetic selection system, where the fusion protein is expressed from pExp and the hybrid targets sites are cloned onto the pTox plasmid harboring the ccdB gyrase toxin;
  • Figure 3 shows chimeric GIY-YIG endonuclease target specificity.
  • Figure 3a is an SDS-PAGE that shows purification of TevN201-zinc finger endonuclease (ZFE).
  • Figure 3b is an SDS-PAGE that shows purification of a BmoN221-ZFE. Lanes are marked as follows: M, marker with molecular weights in kDa indicated on the left; UN, uninduced culture; IND, induced culture; C, crude lysate; FT, flow-through from metal-affinity column; W, wash; E, elution.
  • Figure 3c is a sequencing gel that shows mapping of TevN201-ZFE cleavage sites on the TZ1.33 substrate, with top and bottom cleavage sites indicated below on the Tev-ryA substrate by open and closed triangles, respectively.
  • Figure 3d is a sequencing gel that shows mapping of BmoN221-ZFE cleavage sites on the BZ1.33 substrate, with top and bottom cleavage sites indicated below on the Bmo-ryA substrate.
  • Figure 3e (left) shows the sequences of the wild-type TZ1.33, the TZ1.33 G5A, and TZ 1.33 C IA/G5A mutant substrates and (right) is a bar graph that shows the ECo.smax determinations for each substrate, with ECo.smax values in nM with standard deviations from three experimental trials;
  • Figure 4A provides the amino acid sequences of chimeric GIY-YIG I-
  • FIG. 4B provides the amino acid sequences of chimeric I-Bmol endonucleases of the invention
  • Figure 5 illustrates that TevN201-ZFE functions as a monomer.
  • FIG. 5a (left) is a graph of initial reaction progress for seven TevN201-ZFE concentrations expressed as percent linear product. Protein concentrations from highest to lowest are 47 nM, 32.5 nM, 23 nM, 1 InM, 6nM, 3 nM, and 0.7 nM.
  • Figure 5a (right) is a graph of initial reaction velocity (nM s "1 ) versus TevN201 -ZFE concentration (nM).
  • Figure 5b provides graphs of the results of cleavage assays with 90 nM TevN201 -ZFE and 10 nM one-site pTZ1.31 plasmid (left), or two-site pTZ1.31 plasmids with the same orientation of sites (center) and two-site pTZ1.31 plasmids with the opposite orientation of sites (right);
  • Figure 6 provides a schematic comparison of GIY-YIG ZFEs
  • the GIY-YIG nuclease fusion is to the ryA zinc finger, and (lower) the two ZFNs are fusions of the Fokl nuclease domain to ryA and ryB zinc fingers.
  • the central portion of the GIY- YIG ZFE substrate is shown as random sequence (N).
  • Figure 7 shows various GIY-YIG TAL domain chimeric endonuclease constructs of the invention.
  • Figure 7A (upper) is a schematic of the chimeric endonuclease I-TevI PthXol fusion proteins including amino acid sequences of I- TevI/PthXol fusion proteins, (lower) shows the sequences of various hybrid I- TevI/PthXol substrates.
  • Figure 7B provides the amino acid sequence of various I- TevI/PthXol chimeric endonucleases of the invention.
  • Figure 7C provides the sequences of various I-TevI/PthXol hybrid target sites.
  • Figure 7D shows the amino acid sequences of various I-BmoI/PthXo 1 chimeric endonucleases of the invention.
  • Figure 7E shows the sequences of various I-BmoI PthXol target sites.
  • Figure 8 is photograph of an ethidium bromide gel showing the double-stranded cleavage of various sized substrates.
  • Figure 9 is a schematic of the assay used to individually demonstrate cleavage of top and bottom strands (lower) is a gel showing the results of the assay with variously sized substrates ;
  • Figure 10A is a schematic of an in vitro endonuclease selection protocol.
  • Figure 10B is a graph illustrating the frequency of each nucleotide at various positions in a substrate space as determined by the assay of Figure 10 A.
  • a positive value means an increase in nucleotide frequency, while a negative value means a decrease in nucleotide frequency.
  • position 15 can be mutated without effect on activity.
  • Figure IOC is a schematic showing a correlation of the sequence of the DNA spacer binding motif with the 1-Tevl binding domain. The figure shows a correlation between the preferred DNA bases in the DNA spacer region of the substrate with conserved DNA bases of the native I-Tevl target site in thymidylate synthase genes.
  • Homing endonucleases such as I-Tevl, target genes that encode for conserved proteins. Doing so maximizes their opportunity to spread between related genomes. Further, the homing endonucleases target DNA sequence that corresponds to conserved amino acids of the target gene - again, by using these DNA sequences as recognition determinants it maximizes potential to spread. This figure was using this correlation as a justification for why those positions in the DNA spacer are important;
  • Figure 1 1 graphically illustrates the frequency of the I-Tevl cleavage motif in human cDNAs
  • Figure 12A provides the sequences of the target substrates isolated from a bacterial two plasmid genetic selection assay, and 12B is a bar graph showing percent survival based on substrate spacers as determined by the assay;
  • Figure 13 graphically illustrates the results of a yeast assay for a
  • Substrate TO20 has the following sequence 5'-
  • Substrate Zif268 has the following sequence 5'-GCGTGGGCG-3' (SEQ ID NO:3);
  • Figure 14 graphically illustrates the results of a yeast assay for a
  • Figure 15A provides the amino acid sequence of endonuclease I-Bmol.
  • Figure I 5B provides the amino acid sequence of endonuclease I-Tevl.
  • Figure 15C provides the amino acid sequence of endonuclease I-Tulal.
  • Figure 15D provides an amino acid alignment of the linker regions of I-Tulal, I-Tevl, and I-Bmol;
  • Figure 16A provides the amino acid sequences of DNA binding proteins, PthXo l , AvrBs3, r A, ryB and I-Onul.
  • Figure 16B provides the sequences of the binding sites of each;
  • Figure 17A provides the amino acid sequences of various I-Tevl-zinc finger chimeric endonucleases.
  • Figure 17B provides the amino acid sequences of various I-BmoI-zinc finger chimeric endonucleases;
  • Figure 18 provides the amino acid sequences of I-Tevl-I-Onul chimeric endonucleases
  • Figure 19 provides the amino acid sequences of I-TevI-TAL chimeric endonucleases
  • Figure 20 provides the amino acid sequence of an I-Tulal-ONU chimeric endonuclease
  • Figure 21 provides a sequence alignment of two TAL-effector proteins
  • Figure 22 A provides a general schematic of the preparation of I-TevI -
  • TAL fusions and Figure 22B provides the nucleic acid sequence of the DNA substrates that were used to test the activity of the fusions.
  • Figure 23 shows the results of activity assays of the TEV-TAL fusions against substrates with various length DNA spacer lengths.
  • Figure 24 shows the activity of TEV-TAL12 fusion on various DNA substrates derived from phage thymidylate synthase genes tested in a yeast-based assay system.
  • Figure 25 shows the activity of the Tev-TALl l and Tev-TAL12 fusions on different DNA substrates in HE 293 cells using a GFP assay.
  • Upper panel shows bright field images (left side) and fluorescent images (right side) showing that each construct was active in HE 293 cells as judged by GFP + cells in fluorescent images.
  • Bottom panel shows Western blot analyses of whole cell extracts for full-length GFP.
  • Figure 26 shows the results of assays of optimizing mTALEN architecture in yeast.
  • A Boxplots of ⁇ -galactosidase activity on substrates with different length DMA spacers normalized to a homodimeric ZFN control. Experiments were carried out using the constructs depicted in Figure 22.
  • the fusion points of the I-Tevl S206 fragment to the PthXol N-terminal residue are indicated above each set of plots.
  • the upper and lower limits of the boxes indicate the 25 th and 75 th percentile of the data, the solid bar indicates the median of the data, and the ends of the whiskers represent 1.5 times the interquartile range. Data points outside of the interquartile range (outliers) are shown as black points.
  • Figure 27 shows mapping of mTALEN cleavage sites.
  • A Schematic of double-strand oligonucleotide substrate labeled on top- and bottom-strands. The top-strand nicking product is indicated by an open triangle, and the bottom-strand nicking product by a filled triangle. Representative denaturing polyacrylamide gel of cleavage reactions with the S206-T120 mTALEN. Top- and bottom-strand products are represented by open and filled triangles, respectively.
  • Run-off sequencing reactions allow the determination of cleavage sites, where the complement of the sequence shown in the trace is read (taking into account that an extra "A” is added during the sequencing reactions).
  • C In vitro c leavage mapping on « ?/-PthXol plasmid su bstrates that contain four CNNNG motifs. The open and filled red triangles indicate secondary cleavage sites inferred from run-off sequencing. The electropherograms shown are derived from the nptH substrate.
  • Figure 28 shows the nucleotide preferences between the C and G bases at the CNNNG cleavage site.
  • A Schematic of the substrate used, with the randomized positions indicated and wild-type sequence shown.
  • B Effect of single, double, or triple substitutions in the NNN motif on cleavage efficiency relative to the wild-type AAC sequence. Boxplots are as in Figure 26, with outliers shown as dots.
  • C Heatmap indicating 169-T120 mTALEN activity on individual NNN sequences, grouped according to the number of changes from the wild-type sequence. Axes are labeled by the first, second, and third nucleotide in the NNN sequence.
  • each motif represents the median value plotted on a log 10 scale for N169-T129 mTALEN activity on each sequence.
  • D Boxplots showing the effect of mutations on cleavage activity in all different contexts relative to the wild type motif for each position of the NNN triplet.
  • Figure 29 shows mTALEN accommodation of nucleotide variation in the DNA spacer region.
  • A Boxplot of activity for 45 single nucleotide substitutions in the TP 15 DNA spacer, normalized to mTALEN activity on the TP 15 wild-type substrate. Plotted are the mean activity values of three biological replicates, with each biological replicate averaged from three technical replicates. The wild-type nucleotide at each position in the spacer is indicated at the top of the plot.
  • B On the left are substrates derived from phage-encoded td genes, highlighting differences in the DNA spacer and cleavage motif relative to the wild-type td sequence from phage T4 (lower case red letters). On the right are boxplote showing ⁇ -galactosidase activity in the yeast-based assay for the N169-T120 mTALEN against the different td substrates. Boxplots are labeled as in Figure 26.
  • Figure 30 shows the results of screening of a randomized DNA spacer library in yeast.
  • A Schematic of the TP_1 N library as compared to the wild-type TP 15 sequence, with positions in the DNA spacer number from 1 to 15. Shown below is a representative example of 96-well microtitre plate assay, where the individual wells are colored according to ⁇ -galactosidase activity (in Miller units). The red rectangles at the top right indicate the positive and negative controls. Yellow rectangles indicate active clones whose activity were greater than or within 2 standard deviations of the wild-type control, averaged over three technical replicates.
  • B Plot of nucleotide enrichment per position based on sequencing data for the active and inactive clones.
  • Figure 31 shows activity of mTALENs in HEK293T cells on episomal targets.
  • A Schematic of the vectors used for co-transfection experiments.
  • the mTALEN gene is separated from the mCherry translation reporter by a T2A peptide.
  • (B) Example of mTALEN expression vector transfection efficiency and expression in HEK293T cells, with bright field image on the left and the epifluorescent image (1 sec exposure) of the same field of view on the right.
  • (C) Schematic of the TP15 target, with the Ddel restriction site indicated and sizes of Ddel digestion products indicated.
  • (D) Agarose gel of a representative assay where the target region has been amplified by PCR from total DNA isolated 48 hrs post transfection. Products were digested with Ddel (+) or incubated in buffer without Ddel (-); fragments resistant to cleavage by Ddel due to mutagenic repair are indicated (CR, cleavage resistant).
  • Figure 32 provides the amino acid sequences of some mTALEN constructs of the present invention.
  • a "chimeric" polypeptide typically comprises two or more regions of amino acid sequence (also referred to as domains) that were derived from different proteins.
  • a chimeric polypeptide may also be referred to as a "fusion,” "fusion protein” or “fusion polypeptide.”
  • a chimeric polypeptide of the invention may comprise a first region derived from a first protein and a second region derived from a second protein.
  • the first and the second protein will be different protein molecules, however, the present invention encompasses situations where the first and second regions are portions of one larger protein. Regions of a chimeric polypeptide of the invention may be fused together. As used herein, regions are "fused" when the regions are part of one contiguous string of amino acids.
  • a chimeric polypeptide of the invention is a chimeric polypeptide comprising a first functional activity region and a second functional activity region.
  • functional activity encompasses all types of activities known to those skilled in the art. Examples of functional activities include, but are not limited to, enzymatic activities (e.g., nuclease activity, methylase activity, protease activity, etc), transcriptional regulatory activities (e.g., activation or repression of transcription), cellular localization activities (e.g., nuclear localization signals, cellular compartment localization signals (e.g., chloroplasts)), and binding activities (e.g., DNA binding activities, protein binding activities, etc).
  • the DNA binding activity may be specific to all or a portion of a DNA target sequence and the DNA binding activity may be referred to as DNA-targeting activity.
  • chimeric polypeptides of the invention comprise at least one functional activity region fused to a region of DNA-targeting activity.
  • the regions may be oriented in any order, for example, a region of functional activity may be located N-terminal to the region of DNA-targeting activity or the region of functional activity may be located C-terminal to the region of DNA-targeting activity.
  • Chimeric polypeptides of the invention may comprise a plurality of functional activity regions, at least one of which is a DNA-targeting region. In embodiments of this type, the functional activity regions may be arranged in any order with respect to each other and with respect to the DNA-targeting region.
  • the functional activity regions may be located 1) both N-terminal to the DNA- targeting region, 2) both C-terminal to the DNA-targeting region, or 3) one N- terminal and one C-terminal to the DNA-targeting region.
  • any functional activity region that can be fused to a DNA- targeting region and retain activity can be used in the practice of the present invention.
  • a functional activity region may comprise a nuclease activity. Any nuclease activity known to those skilled in the art may be used in the practice of the present invention. Any protein having nuclease activity may be used as a source of the functional activity regions in the chimeric polypeptides of the invention. [0057] In general, any nuclease, or domain of a nuclease that has nuclease activity, that can be fused to a DNA-targeting region and retain the nuclease activity can be used in the practice of the present invention. In some embodiments, the nuclease may function as a monomer.
  • any site specific nuclease that is functional as a monomer can be used as the source of the nuclease domain for use in the present invention.
  • the nuclease domain is derived from a homing endonuclease, for example, a homing endonuclease of the GIY-YIG family of homing endonucleases.
  • site specific nucleases that cleave double- stranded DNA as monomers include, but are not limited to, Mspl, HinPlI, Mval and Bcnl.
  • the present chimeric GIY-YIG endonuclease may comprise a GIY-
  • the GIY-YIG nuclease domain is an ⁇ / ⁇ structure comprising at least about 90-100 amino acids, the amino acid sequence -GIY- spaced from the amino acid sequence -YIG- by 10-1 1 amino acids which forms part of a three- stranded antiparallel ⁇ -sheet.
  • Residues that may be important for nuclease activity include a glycine residue within the GIY- YIG motif, an arginine residue about 8-10 residues downstream of the --GIY- sequence (e.g.
  • GIY-YIG nuclease domains include, but are not limited to, the nuclease portion of I-Bmol (for example, residues 1-92), the full-length amino acid sequence of which is illustrated in Fig. 15A, I-Tevl (for example, at least residues 1 -1 14), the full-length sequence of which is illustrated in Fig. 15B, and I-Tulal (for example, residues 1 -1 14), the full-length sequence of which is illustrated in Fig. 15C.
  • GIY-YIG nuclease domains may also be utilized within the present chimeric endonuclease.
  • the term “functionally equivalent” refers to variant nuclease domains which vary from a wild-type or endogenous sequence but which retain nuclease function, even though it may be to a lesser degree. Accordingly, variant GIY-YIG nuclease domains may include one or more amino acid substitutions, deletions or insertions at positions which do not eliminate nuclease activity.
  • Variant nuclease domains may comprise at least about 50% sequence similarity with a native nuclease sequence, at least about 60-70%», or at least about 80%-90% or greater sequence similarity with a native nuclease sequence, to retain sufficient nuclease activity.
  • variant GIY-YIG nuclease domains include N- or C- terminal truncated GIY-YIG nuclease domains, for example, N-terminal truncations of up to about 20 amino acid residues and C-terminal truncations of up to about 15 amino acid residues, and one or more amino acid substitutions, insertions or deletions which do not adversely affect nuclease activity, for example within the N-terminus up to about the amino acid at position 20 or within the C-terminus from about the amino acid at position 75, and amino acid substitutions within the 10-1 1 amino acid spacer between -GIY- and -YIG-.
  • suitable amino acid substitutions include conservative amino acid substitutions, for example, substitution of an amino acid with a hydrophobic side chain with a like amino acid, e.g. alanine, valine, leucine, isoleucine, phenylalanine and tyrosine; substitution of an amino acid with an uncharged polar sidechain with a like amino acid, e.g. serine, threonine, asparagine and glutamine; substitution of an amino acid having a positively charged sidechain with a like amino acid, e.g. arginine, histidine and lysine; or substitution of an amino acid having a negatively charged sidechain with a like amino acid, e.g. aspartic and glutamic acid.
  • Variant GIY-YIG nuclease domains may also include one or more modified amino acids, for example, amino acids including modified sidechain entities which do not adversely affect nuclease activity.
  • nuclease domains derived from: other homing endonucleases, for example, the HNH family of homing endonucleases; restriction enzymes (including other Type IIS enzymes with properties distinct from Fokl); or DNA repair nucleases may be used in the practice of the present invention.
  • Nucleases or nuclease domains for use in the present invention typically function as a monomer.
  • the nuclease or nuclease domain will make a double-strand break in DNA.
  • a nuclease or nuclease domain may only cleave one strand, i.e., may nick one strand of DNA. Such nickases have been shown to induce recombination and gene knockouts in mammalian cells with reduced levels of toxicity relative to double-strand nucleases.
  • a nuclease or nuclease domain for use in the present invention will have at least a minimal amount of site-specificity.
  • nuclease or nuclease domain will be entirely not site specific. This will allow the greatest flexibility in the application of the chimeric polypeptides of the invention.
  • a nuclease or nuclease domain may be derived from an HNH family endonucleases.
  • HNH endonucleases have a two-domain structure similar to GIY-YIG homing endonucleases.
  • the catalytic nuclease domain is located in the N-terminal region of the polypeptide and comprises a catalytic domain defined by the amino acid motif HNH.
  • the C-terminal portion of the polypeptide comprises the DNA-binding domain.
  • HNH endonucleases usually function as nickases (ie. nick one strand of DNA).
  • the nuclease or nuclease domain for use in the present invention may be derived from the HNH enzyme I-Hmul (Accession:P34081. lGI:465641) the sequence of which is specifically incorporated herein by reference.
  • I-Hmul looks structurally very similar to GIY-YIG enzymes.
  • the HNH domain from I-Hmul can be fused to a DNA- targeting domain to create a targeted ickase enzyme.
  • I-Hmul has sequence specificity at the nicking site. In some embodiments, the specificity of cleavage may be engineered out by altering one or more of the amino acids involved in DNA contact.
  • Such amino acids are known based on a co-crystal of I-Hmul with DNA (Shen et al. JMB 342:43-56, 2004).
  • Other HNH family enzymes may be used as a source of a nuclease domain in the practice of the present invention.
  • Other examples of suitable sources of nuclease activity include colicins that degrade DNA.
  • a nuclease region may be derived from other
  • Type IIS enzymes On suitable example is Eco31I (Accession:AAM09638.2, GI:56788324), This is a type IIS enzyme, similar to Fokl. As all Type IIS restriction enzymes, Eco31 I binds a DNA site, but cleaves at a distance from the binding site. Eco311 has a similar domain structure to Fokl, but the C-terminal cleavage domain contains an HNH motif. Interestingly, the enzyme functions as a monomer and makes a double-strand break. (Jakubauskas et al. Biochemistry 47:8546, 2008). Other type IIS enzymes are known to those skilled in the art and may be used in the practice of the present invention.
  • Non-specific nucleases may also be used in the practice of the invention. Examples include Staphylococcal nuclease. Zinc finger fusions with Staph nuclease have been prepared that that are active (Mineta et al. Biochemistry 47: 12257, 2008).
  • DNA-repair nucleases may be used in the practice of the invention.
  • DNA repair enzymes that nick or make double-stranded breaks (DSBs) in DNA, usually at a site of DNA damage (e.g., an abasic site), or at a specific DNA structure (e.g., cruciform DNA).
  • a site of DNA damage e.g., an abasic site
  • a specific DNA structure e.g., cruciform DNA.
  • the nuclease domain from these types of nucleases may be used in the practice of the present invention.
  • Suitable examples include, but are not limited to, UvrC (Accession:ZP_03002418.1, GI: 18849 148) which has a GIY-YIG domain that nicks one strand, MutL ( Accession :P23367.2G1: 127552) a bacterial enzyme and its human homolog PMS2 (Accession:NP_000526.1 , GI:4505913).
  • AP endonucleases There are many examples of DNA repair enzymes that recognize DNA mismatches. The endonuclease activity of such enzymes can be used as a source of nuclease activity for the practice of the present invention.
  • Minimal nuclease domains may be used as the nuclease activity in the practice of the present invention. Using crystal structures and domain structure studies as a guide, the boundaries of the region of the enzyme having nuclease activity can be identified. Once identified, the region can be cloned using standard techniques and can serve as a nuclease activity.
  • Nucleases for use in the present invention may be from any source, for example, archaebacteria, bacteria, viruses, eukaryotes, organelles (e.g., mitochondria and chloroplast) of eukaryotes, plants, algae, fungi, or protozoa. Any source may be used so long as the nuclease activity is functional in the desired target organism, for example, in a eukaryotic cell (e.g., a mammalian cell, a plant cell, etc.).
  • a eukaryotic cell e.g., a mammalian cell, a plant cell, etc.
  • a functional activity region may comprise a
  • DNA-modifying activity for example, a DNA methylase activity or a cytosine deaminase activity.
  • a functional activity region that may be used in the practice of the present invention is a functional activity region having DNA methylase activity.
  • Any DNA methylase activity known to those skilled in the art may be used in the present inventions. Examples include, but are not limited to, methylase activity that generates N6-methyladenine (EC 2.1.1.72), methylase activity that generates N4- methylcytosine (EC 2.1.1.1 13), and methylase activity that generates C5- methylcytosine (EC 2.1.1.37).
  • Any protein having DNA methylase activity may be used as a source of the functional activity regions in the chimeric polypeptides of the invention.
  • Examples include DNA (cytosine-5) methyltransferase I (Accession NP 001 124295.1 , GI: 195927037); DNA (cytosine-5)-methyltransferase 3A isoform a (Accession:NP_072046.2, GI: 12751473), DNA (cytosine-5)-methyltransferase 3A isoform b (Accession:NP_715640.2, GI:77176455); and DNA (cytosine-5 -)- methyltransferase 3 beta (Accession:AAD53062.1 , GI:5823166), the sequences of which are specifically incorporated herein by reference.
  • the DNA modifying activity that may be used in the practice of the invention is cytosine deaminase activity (E.C. 3.5.4.1). These could be useful for making C:G -> T:A transitions in DNA at specific sites.
  • Any protein having cytosine deaminase activity may be used as a source of the functional activity regions in the chimeric polypeptides of the invention. Examples include E. coli cytosine deaminase (Accession:BAE761 19.1, GI:85674479) and yeast cytosine deaminase (Accession : A AB67713.1, GI:2343114), the sequences of which are specifically incorporated herein by reference.
  • a functional activity region may comprise a transcription regulatory activity.
  • the activity may be a transcription activation activity. Any transcription activation activity known to those skilled in the art may be used in the practice of the present invention. Any protein having transcription activation activity may be used as a source of the functional activity regions in the chimeric polypeptides of the invention.
  • HSV VP16 (Accession:AAA45864.1, GI:330320), the p65 subunit of human transcription factor NF-kB (Accession:Q04206.2, GI:62906901), Nicotiana tabacum ERF2 (Accession Q40479.1 , GI:57012759), Nicotiana tabacum ERF4 (Accession Q40477.1, GI:57012757), Arabidopsis thaliana ERF1 (Accession P93835.2 GI:47605622), Arabadopsis thaliana ERF2 (Accession O80338.1. GI:7531 108), and Arabadopsis thaiiana ERF5 (Accession:BAA97157.1 , GI:8809606), the sequences of which are specifically incorporated herein by reference.
  • a functional activity region may comprise a transcription repression activity. Any transcription repression activity known to those skilled in the art may be used in the practice of the present invention. Any protein having transcription repression activity may be used as a source of the functional activity regions in the chimeric polypeptides of the invention. Examples include tobacco Nicotiana tabacum ERF3 (Accession Q9SXS8.1, GI:57012880), Arabadopsis thaiiana ERF3 (Accession 080339.1 GI:7531 109), and Arabadopsis thaiiana ERF4 (Accession Q9FJ93.1, GI:47605744), the sequences of which are specifically incorporated herein by reference.
  • Functional activity regions may be linked by linking domains.
  • a nuclease domain may be linked to a DNA-targeting domain via a linking domain.
  • Other functional domains e.g., methylase domains, transcriptional regulatory domains etc, may be linked to a DNA-targeting domain via a linking domain.
  • the GIY-YIG nuclease domain may be linked to a DNA- targeting domain via a linking domain.
  • the linking domain will generally be a polypeptide of a length sufficient to permit the nuclease domain to retain nuclease function when linked to the DNA-targeting domain, and sufficient to permit the DNA-binding domain to bind the endonuclease to a target substrate.
  • the linking domain may be from 1 amino acid residue to about 100 amino acid residues, from about 1 amino acid residue to about 90 amino acid residues, from about 1 amino acid residue to about 80 amino acid residues, from about 1 amino acid residue to about 70, from about 1 to about 60 amino acid residues, from about 1 to about 50 amino acid residues, from about 1 to about 40 amino acid residues, from about 1 to about 30 amino acid residues, or from about 1 amino acid residue to about 25 amino acid residues.
  • the linking domain may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues in length.
  • the length of the linker domain may be adjusted depending on the distance between the binding and cleavage sites on a target nucleic acid molecule.
  • chimeric endonucleases of the invention can cleave nucleic acid molecules where the binding and cleavage sites are separated by varying numbers of basepairs.
  • the linking domain may be a random sequence, for example, may be one or more glycine residues.
  • the linking domain may be a simple repeat of amino acids, for example, GS, which may be repeated multiple times. As used herein, such a repeat will be indicated by placing the amino acids in parenthesis and using a subscript to indicate the number of times repeated.
  • GS indicates a linking domain of four repeats of the amino acids glycine and serine.
  • (G 4 S) 3 indicates three repeats of the sequence G-G-G-G-S.
  • the linker domain may comprise one or more glycine residues in addition to one or more amino acid residues.
  • the linking domain may be from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, or may be 100% glycine.
  • the linking domain may be flexible or may comprise one or more regions of secondary structure that impart rigidity, for example, alpha helix forming sequences.
  • the linking domain may be the endogenous linker associated with the GIY-YIG nuclease, e.g.
  • the linking domain may be unrelated to the nuclease domain, i.e. the I-Tevl linker or portion thereof may be utilized with the I-Bmol or 1-TulaI nuclease regions, or the I-Bmol or I-Tulal linker or portion thereof may be used with the 1-TevI nuclease domain.
  • nuclease-linker portion of an endonuclease may be utilized, such as the I-Tevl nuclease domain and its linker region from about amino acid residue 1 to about amino acid residue 1 14, from about amino acid residue 1 to about amino acid residue 128, from about amino acid residue 1 to about amino acid residue 141 , from about amino acid residue 1 to about amino acid residue 169, from about amino acid residue 1 to about amino acid residue 170, from about amino acid residue 1 to about amino acid residue 201, from about amino acid residue 1 to about amino acid residue 203, from about amino acid residue 1 to about amino acid residue 206; the I-Bmol nuclease domain and linker from about amino acid residue 1 to about amino acid residue 96, from about amino acid residue 1 to about amino acid residue 1 5, from about amino acid residue 1 to about amino acid residue 125, from about amino acid residue 1 to about amino acid residue 139, from about amino acid residue 1 to about amino acid residue 159, from about amino acid residue 1 to about amino acid residue 221,
  • the linking domain may be modified from a wild-type or native linking domain sequence. Suitable modifications include one or more amino acid substitutions, deletions or insertions, that do not impact on the function of the endonuclease, i.e. do not eliminate binding of the DNA- targeting domain to its substrate, nor eliminate nuclease activity.
  • the native I-Tevl linker has some DNA sequence preference. Accordingly, the present invention provides modified I-Tevl linkers wherein the sequence of the native protein linker has been modified to change its DNA binding specificity, without affecting nuclease activity, to broaden or reduce targeting potential based on a specific target DNA sequence.
  • Variant linking domains may comprise linking domain sequence to function effectively as a linking domain. Examples of at least about 50% sequence similarity with a native linking domain sequence, at least about 60-70%, and at least about 80%-90% or greater sequence similarity with a native linking domain to function as an effective linking domain. Suitable modifications include truncation of a native linking domain as set out above, and conservative amino acid substitutions as set out with respect to the nuclease domain.
  • linkers can be designed as needed for particular circumstances in accordance with known techniques (see, for example, Fan Xue et al., "LINKER: a web server to generate peptide sequences with extended conformation", Nucl. Acids Res.
  • linkers can be designed based on specific target sequences, i.e., linkers are designed to have DNA binding activity with a particular sequence(s).
  • Suitable non-specific linkers without DNA binding activity can also be used. Examples of such non-specific linkers include, but are not limited to, linkers that interact non-specifically with the minor groove of DNA.
  • Non-specific linker is a linker having the sequence TG SI RPRAIGGS KPRVAT. This linker interacts with the sugar and phosphate of DNA, i.e., interacts with DNA in a non-specific manner.
  • a person of ordinary skill in the art will recognize that modifications of non-specific linker sequences can be required based on a particular context of use for the linker.
  • the DNA-targeting domain may be any suitable domain that binds
  • DNA-targeting domains include, but are not limited to, the DNA binding domains of TAL-effector proteins, such as PthXol and AvrBs3 (from Xanthamonas campestris); zinc finger domains, e.g. ryA zinc finger binding domain and ryB zinc finger binding domain, and other distinct DNA-binding platforms, such as the binding domain in LAGLIDADG homing endonucleases, e.g. I-Onul, which have reprogrammable DNA-binding specificity similar to zinc fingers or TAL domains.
  • a functionally equivalent variant binding domain based on a native binding domain, i.e.
  • a binding domain which incorporates sequence modifications but which retains DNA binding activity may also be utilized in the present chimeric endonuclease.
  • Variant binding domains may comprise at least about 50% sequence similarity with a native binding domain sequence, at least about 60-70%, and at least about 80%-90% or greater sequence similarity with a native binding domain to retain sufficient binding activity.
  • Such a variant binding domain may include one or more of: an N- or C-terminal truncation, one or more amino acid substitutions, deletions or insertions, or modification of an amino acid, for example, modification of an amino acid sidechain entity.
  • the DNA- targeting domain is typically bound at its N-terminal end to the linking domain or to the nuclease domain.
  • the DNA-targeting domain may be bound at its C-terminal end to the linking domain or to the nuclease domain.
  • One of ordinary skill in the art is capable of using standard techniques to fuse the DNA- targeting domain at either end to a linking domain and/or a nuclease or other functional domain.
  • the targeting specificity of the present chimeric G1Y-YIG endonuclease is a function of DNA-targeting domain and may be modified or enhanced by modifying the specificity of the DNA-targeting domain as set out above. Additionally, for example, the specificity of the 3-zinc fmger DNA-targeting domain of ryA or ryB may be enhanced by addition of zinc fingers to generate a 4-, 5-, or 6- zinc finger fusion protein.
  • the DNA-targeting domain of a chimeric endonuclease is a TAL domain, or a modified TAL domain.
  • suitable TAL domains are known in the art, for example US 201 1/0301073 discloses Novel DNA-Binding Proteins and Uses Thereof and is specifically incorporated herein for its teaching of the structure of the DNA binding domain of TAL-effectors (i.e., TAL domain).
  • a TAL domain is generally comprised of a plurality of repeat units that are typically 33 to 35 amino acid residue long segments and the repeats are typically 90- 100% homologous to each other. Suitable repeats include, but are not limited to, those from Xanthomonas, for example,
  • LTPEQVVAIASNIGG QALETVQALLPVLCQAHG (SEQ ID NO:4), LTPDQ V VAI A SEGGG QALET VQRLLPV LC QAHG (SEQ ID NO:5), and LTPEQVVAIASNIGGKQALETVQRLLPVLCQAHG (SEQ ID NO:6), those from Ralstonia solanacearum, for example,
  • LTPQQWAIASNTGGKRALEAVCVQLPVLRAAPYR (SEQ ID NO:7), LSTEQWAIASN GG QALEAVKAHLLDLLGAPYV (SEQ ID NO:8) and LDTEQVVAIASHNGG QALEAV ADLLDLRGAPYA (SEQ ID NO:9).
  • TAL domain L(T P)(P/Q)(E/A/D/V)QVVAIASHDGGKQAL(E/A)T(V/M)QRLLPVLCQ(A/D)HG (SEQ ID NO: 10).
  • RVD Repeat Variable Diresidue
  • amino acid residues NI correspond to adenine
  • amino acid residues HD correspond to cytosine
  • amino acid residues NG correspond to thymine
  • amino acid residues NN correspond to guanine (and to a lesser degree adenine)
  • amino acid residues NS correspond to A, C, T or G
  • amino acid residues N* (where * indicates a no amino acid residue) correspond to C or T
  • amino acid residues HG correspond to T.
  • RVDs are disclosed in US 201 1/0301073 and are specifically incorporated herein by reference. Using the known DNA sequence of a gene, a chimeric endonuclease of the invention may be constructed specific to any gene locus.
  • suitable gene loci include, but are not limited to, NTF3, VEGF, CCR5, IL2Ry, BAX, BA , FUT8, GR, DHFR, CXCR4, GS, Rosa26, AAVS 1 (PPP1 R1 2C), MHC genes, P1TX3, ben-1, Pou5 F 1, (OCT4), CI, RPD1 , and any other genes known to those skilled in the art.
  • a TAL domain may be constructed by fusing a plurality of repeat units. Any number of repeat units may fused to create a TAL domain, for example, from about 5 repeat units to about 30 repeat units, from about 5 repeat units to about 25 repeat units, from about 5 repeat units to about 20 repeat units, from about 5 repeat units to about 1 repeat units, or from about 5 repeat units to about 10 repeat units, from about 7.5 repeat units to about 30 repeat units, from about 7.5 repeat units to about 25 repeat units, from about 7.5 repeat units to about 20 repeat units, from about 7.5 repeat units to about 15 repeat units, or from about 7.5 repeat units to about 10 repeat units.
  • a TAL domain of the invention may comprise
  • any two repeat units in a given TAL domain may be from about 75% to about 100%, from about 80% to about 100%, from about 85% to about 100%, from about 90% to about 100%, from about 91% to about 100%, from about 92% to about 100%, from about 93% to about 100%, from about 94% to about 100%, from about 95% to about 100%, from about 96% to about 100%, from about 97% to about 100%, from about 98% to about 100%, or from about 99% to about 100% , from about 75% to about 95%, from about 80% to about 95%, from about 91 % to about 95%, from about 92% to about 95%, from about 93% to about 95%, from about 75% to about 90%, from about 80% to about 90%, from about 82% to about 90%, from about 84% to about 90%, from about 86% to about 90%, or from about 8
  • TAL domains of the invention may also comprise one or more half repeats that are typically on either the N-terminal, the C-terminal, or on both the island C-terminals of the TAL domain.
  • at least one repeat unit is modified at some or all of the amino acids at positions 4, 11, 12, 13 or 32 within the repeat unit.
  • at least one repeat unit is modified at 1 or more of the amino acids at positions 2, 3, 4, 1 1 , 12, 13, 21, 23, 24, 25, 26, 27, 28, 30, 31,32, 33, 34, or 35 within one repeat unit.
  • TAL domains can be constructed to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the hypervariable diresidue region, for example positions 12 and/or 13 of a repeat unit within a TAL protein.
  • the amino acids at positions 4, 1 1, and 32 can also be engineered.
  • a typical RVDs can also be selected for use in an engineered TAL protein, enabling specification of a wider range of non-natural target sites.
  • a NK RVD can be selected for use in recognizing a G nucleotide in the target sequence.
  • Amino acids in the repeat unit can be altered to change the characteristics (i.e., stability or secondary structure) of the repeat unit.
  • Engineered TAL proteins can be proteins that are non-natural ly occurring.
  • the genes encoding TAL repeat domains can be engineered at the DNA level such that the codons specifying the TAL repeat amino acids are altered, but the specified amino acids are not (e.g., via known techniques of codon optimization).
  • Examples of engineered TAL proteins include, but are not limited to, those obtained by design and/or selection.
  • a designed TAL protein can be a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design can include application of substitution rules and computerized algorithms for processing information in a database storing information of existing TAL designs and binding data.
  • a selected TAL domain can be a non-naturally occurring or atypical domain whose production results primarily from an empirical process such as phage display, interaction trap, or hybrid selection.
  • TAL domains can be derived from any suitable TAL protein.
  • TAL proteins include, but are not limited to, TAL proteins derived from Ralstonia spp. or Xanthamonas spp..
  • the DNA-targeting domain can comprise one or more one or more naturally occurring and/or engineered TAL domain units derived from the plant pathogen Xanthomonas.
  • the DNA-targeting domain can comprise one or more naturally occurring and/or engineered TAL domain units derived from the plant pathogen Ralstonia solanacearum, or other TAL DNA binding domain from the TAL protein family.
  • the TAL DNA binding domains as described herein can include (i) one or more TAL repeat units not found in nature; (ii) one or more naturally occurring TAL repeat units; (iii) one or more TAL repeat units with atypical RVDs; and combinations of (i), (ii) and/or (iii).
  • a TAL DNA binding domain as described herein can consist of completely non- naturally occurring or atypical repeat units.
  • the TAL domain units naturally occurring or engineered
  • the target sites useful can be subject to evaluation by other criteria or can be used directly for design or selection (if needed) and production of a TAL- fusion protein specific for such a site.
  • a further criterion for evaluating potential target sites can be their proximity to particular regions within a gene. Target sites can be selected that do not necessarily include or overlap segments of demonstrable biological significance with target genes, such as regulatory sequences. Additional criteria for further evaluating target segments can include prior availability of TAL- fusion proteins binding to such segments or related segments, and/or ease of designing new TAL- fusion proteins to bind a given target segment.
  • a TAL- fusion protein that binds to the segment can be provided by a variety of approaches. Once a TAL-fusion protein has been selected, designed, or otherwise provided to a given target segment, the TAL-fusion protein or the DNA encoding can be synthesized. The TAL-fusion protein or a polynucleotide encoding it can then be used for modulation of expression, or analysis of the target gene containing the target site to which the TAL-fusion protein binds.
  • Suitable modified TAL domains may include one or more amino acid deletions, insertions or substitutions which do not eliminate the DNA binding activity thereof, for example, modifications at one or more amino acid residues other than amino acid residues at position 12 and 13, such as those indicated with multiple amino acid residues in parenthesis in the above sequence.
  • Other proteins having TAL domains can be used to identify suitable repeats that can be used to construct a DNA- targeting domaim. Examples include, but are not limited to, Avrb6 from Xanthomonas citri subsp.
  • Oryzae GenBank accession number AAN01357.1 AvrXa5 from Xanthomonas oryzae pv. Oryzae GenBank accession number AAQ79773.2, PthXo3 from Xanthomonas oryzae pv. Oryzae GenBank accession number AAS46027.1, and PthXo4 from Xanthomonas oryzae pv. Oryzae GenBank accession number AAS58127.2. Additional examples include, but are not limited to, PthB from Xanthomonas xonopodis pv.
  • Oryzae GenBank accession number AAS58130.3 avirulence protein AvrXa7-2M from Xanthomonas oryzae pv.
  • Oryzae GenBank accession number AAT46123.1 avirulence protein AvrXa7-3M from Xanthomonas oryzae pv.
  • Oryzae GenBank accession number AAT46124.1 avirulence protein AvrXa7-3M from Xanthomonas oryzae pv.
  • Oryzae GenBank accession number AAT46124.1 Avr/Pthl 3 from Xanthomonas oryzae pv.
  • Oryzicola GenBank accession number AAW59491.1 Avr/Pth3 from Xanthomonas oryzae pv.
  • Oryzicola GenBank accession number AAW59492.1 Avr/Pthl4 from Xanthomonas oryzae pv. Oryzicola GenBank accession number AAW59493.1, avirulence protein from Xanthomonas oryzae pv. Oryzae KACC 10331 GenBank accession number AAW77510.1, Hax2 from Xanthomonas campestris pv. Armoraciae GenBank accession number AAY43358, Hax3 from Xanthomonas campestris pv. Armoraciae GenBank accession number AAY43359.1 , Hax4 from Xanthomonas campestris pv.
  • Armoraciae GenBank accession number AAY43360.1 R19.5 from Xanthomonas oryzae pv. Oryzae GenBank accession number AAY54166.1, AvrXa27 from Xanthomonas oryzae pv. Oryzae GenBank accession number AAY54168.1, R13.5 from Xanthomonas oryzae pv. Oryzae GenBank accession number AAY54169.1, R23.5 from Xanthomonas oryzae pv. Oryzae GenBank accession number AAY54170.1 , PthXo7 from Xanthomonas oryzae pv.
  • Oryzae GenBank accession number ABB70129.1 PthXo6 from Xanthomonas oryzae pv. Oryzae GenBank accession number ABB70183.1, and PthAW from Xanthomonas citri pv. Citri GenBank accession number AB077779.1. The sequence of each of these proteins is specifically incorporated herein by reference.
  • Proteins from Ralstonia solanacearum having TAL domains can be used to identify suitable repeats that can be used to construct a DNA-targeting domain. Examples include, but are not limited to, AvrBs3-like effector from Ralstonia solanacearum GenBank accession number ABO27067.1 , AvrBs3-like effector from Ralstonia solanacearum GenBank accession number ABO27068.1, AvrBs3-like effector from Ralstonia solanacearum GenBank accession number ABO27069.1, AvrBs3-like effector from Ralstonia solanacearum GenBank accession number ABO27070.1 , AvrBs3-like effector from Ralstonia solanacearum GenBank accession number ABO27071.1 , and AvrBs3-like effector from Ralstonia solanacearum GenBank accession number ABO27072.1.
  • the DNA-targeting domain can be constructed to target non-nuclear
  • TAL-effectors can include a nuclear localization sequence (NLS) for localization to a eukaryotic nucleus.
  • NLS nuclear localization sequence
  • a TAL-effector protein can be constructed to include a localization sequence for targeting cellular components other than the nucleus.
  • a TAL-effector can be fused to a targeting sequence that targets mitochondria and/or chloroplasts. Use of this targeting domain can allow modification of a mitochondrial genome or a plastid genome.
  • a TAL domain of the invention may also comprise flanking sequences at the N- and/or C-terminal of the TAL domain.
  • the flanking sequences may be of any length that does not interfere with the DNA-binding of the TAL domain.
  • Flanking sequences may be from about 1 amino acid residue to about 300 amino acid residues, from about 1 amino acid residue to about 250 amino acid residues, from about 1 amino acid residue to about 200 amino acid residues, from about 1 amino acid residue to about 150 amino acid residues, from about 1 amino acid residue to about 125 amino acid residues, from about 1 amino acid residue to about 100 amino acid residues, from about 1 amino acid residue to about 75 amino acid residues, from about 1 amino acid residue to about 50 amino acid residues, from about 1 amino acid residue to about 40 amino acid residues, from about 1 amino acid residue to about 30 amino acid residues, from about 1 amino acid residue to about 20 amino acid residues, or from about 1 amino acid residue to about 10 amino acid residues.
  • the flanking sequences may be of any amino acid sequence.
  • flanking sequences may be derived from the naturally occurring sequence of a TAL-effector protein, which may be the same or different TAL-effector protein from which the repeat units are derived.
  • the present invention encompasses TAL domains comprising repeat units having an amino acid sequence found in a first TAL-effector protein and one or more flanking sequences found in a second TAL-effector protein.
  • One suitable source for flanking sequences is amino acid residues 130 to 416 of SEQ ID NO: 101 which is the N- terminal flanking region of PthXol ( Figure 7A).
  • a flanking sequence may comprise all or a part of amino acid residues 130 to 416 of SEQ ID NO: 101.
  • a flanking sequence may comprise from about amino acid residue 150 to about amino acid residue 416, from about amino acid residue 175 to about amino acid residue 416, from about amino acid residue 200 to about amino acid residue 416, from about amino acid residue 225 to about amino acid residue 416, from about amino acid residue 250 to about amino acid residue 416, from about amino acid residue 275 to about amino acid residue 416, from about amino acid residue 300 to about amino acid residue 416, from about amino acid residue 325 to about amino acid residue 416, from about amino acid residue 350 to about amino acid residue 416, from about amino acid residue 375 to about amino acid residue 416, or from about amino acid residue 400 to about amino acid residue 416.
  • a flanking sequence may have sequence identity with one or more of the flanking sequence above.
  • a flanking sequence may comprise a sequence that is from about 80% to about 100% identical to the sequence of from about amino acid residue 350 to about amino acid residue 416, from about 85% to about 100% identical, from about 90% to about 100% identical, from about 95% to about 100% identical, from about 80% to about 95% identical, from about 80% to about 90% identical, or from about 80% identical to about 85% identical.
  • a flanking sequence may comprise a sequence that is from about 80% to about 100% identical to the sequence of from about amino acid residue 300 to about amino acid residue 416, from about 85% to about 100% identical, from about 90% to about 100% identical, from about 95% to about 100% identical, from about 80% to about 95% identical, from about 80% to about 90% identical, or from about 80% identical to about 85% identical.
  • a flanking sequence may comprise a sequence that is from about 80% to about 100% identical to the sequence of from about amino acid residue 250 to about amino acid residue 416, from about 85% to about 100% identical, from about 90% to about 100% identical, from about 95% to about 100% identical, from about 80% to about 95% identical, from about 80% to about 90% identical, or from about 80% identical to about 85% identical.
  • Chimeric endonucleases of the invention may optionally comprise one or more functional domains.
  • Suitable functional domains include, but are not limited to, transcription factor domains (activators, repressors, co-activators, co-repressors) , additional nuclease domains, silencer domains, oncogene domains (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members etc.); DNA repair enzymes and their associated factors and modifiers; DNA rearrangement enzymes and their associated factors and modifiers; chromatin associated proteins and their modifiers (e.g.
  • kinases e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases
  • DNA targeting enzymes such as transposons, integrases, recombinases and resolvases and their associated factors and modifiers, nuclear hormone receptors, and ligand binding domains.
  • Chimeric polypeptides of the invention can include further modifications to control activity in various cell types.
  • a chimeric polypeptide can be fused to a sequence that promotes degradation.
  • sequences include, but are not limited to, a DEATH domain sequence, a sequence known to be ubiquitinated to promote degradation, a protein kinase sequence that when phosphorylated promotes degradation.
  • a further example is to construct a chimeric polypeptide having a self-splicing intein in the middle.
  • An intein is a protein segment that can excise itself and rejoin the remaining protein portions with a peptide bond during protein splicing.
  • Inteins can be engineered to be redox sensitive (as described in, for example, Callahan BP et al., "Structure of catalytically competent intein caught in a redox trap with functional and evolutionary implications", Nat. Struct. ol. Biol., 18(5) 630-3.)
  • redox conditions in the cell can control splicing of the intein and ligation of the N- and C-terminal domains of the chimeric polypeptide.
  • a similar example is to insert an intron into the TAL coding region, where the intron can be spliced under certain cellular conditions and/or in specific cell types.
  • a chimeric polypeptide of the invention may comprise one or more nuclear localization signals or other amino acid sequences that direct the distribution of the c himeric polypeptide in cell.
  • Suitable nuclear localization sequences are known in the art. Examples include, but are not limited to, the nucleoplasmin NLS RX, 0 KK L (SEQ ID NO: 1 1 ) (Moore JD,J Cell Biol.
  • a chimeric polypeptide of the invention may comprise one or more cellular localization domains.
  • a cellular localization domain may be a transit peptide, e.g., a chloroplast transit peptide. Suitable chloroplast transit peptides are known in the art.
  • the transit peptide may be an algal chloroplast transit peptide. Sutable examples include, but are not limited to, those from Chlamydomonas reinhardtii disclsoed in Franzen FEBS 260: 165- 168, 1990 the sequences of which are specifically incorporated herein by reference.
  • the present invention provides novel chimeric endonucleases that can be engineered to cleave virtually any nucleic acid molecule at a desired site. This is accomplished by selecting the desired binding and cleaving domains and using recombinant DNA techniques to construct a fusion protein comprising the selected domains.
  • chimeric endonucleases invention are capable of creating double-stranded breaks in DNA molecule, for example, in the genome of an organism. Double-stranded breaks thus created may be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination at a predetermined chromosomal locus.
  • a novel chimeric endonuclease comprising a GIY-YIG nuclease domain which is linked to a DNA- targeting domain by a linking domain.
  • chimeric endonucleases of the prior art for example, TALENs comprising the Fokl nuclease domain
  • chimeric endonucleases of the present invention are capable of cleaving DNA as monomers. This allows greater flexibility in construction and ease in use as compared to the chimeric endonucleases of the prior art. Chimeric endonucleases of the invention will be particularly useful for in vivo applications as they do not require dimerization in situ to be effective.
  • chimeric endonucleases include, but are not limited to,
  • Tevl nuclease linked to PthXol TAL DNA targeting domain I-Tevl nuclease linked to ryA or ryB zinc finger DNA targeting domain, I-Tevl nuclease linked to Onul DNA targeting domain, I-Bmol nuclease linked to PthXol TAL DNA targeting domain, I-Bmol nuclease linked to ryA or ryB zinc finger DNA targeting domain, I- Tulal linked to ryA or ryB zinc finger DNA targeting domain, Tula linked to a PthXol TAL DNA-targeting domain, and Tula linked to the I-Onul targeting domain.
  • Nucleases may be linked via a linking domain as described above, either the linking domain native to the nuclease or derived from the linking domain native to the nuclease, or a linking domain of a different nuclease or derived from a different nuclease, or a linking domain comprising a random sequence.
  • An engineered TAL-effector protein and TAL-effector fusion protein can have a novel binding specificity, compared to a naturally-occurring TAL-effector protein.
  • Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising nucleotide sequences for modules for single or multiple TAL-effector repeats.
  • Exemplary selection methods including phage display and two-hybrid systems, are disclosed in US Patents 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6, 140,466; 6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB 2,338,237.
  • TALE related domains containing all possible mono- and dipeptide sequences have been constructed and assembled into candidate TAL-effector proteins.
  • one or more TAL- effector repeat units of the DNA-binding protein comprise atypical RVDs.
  • the repeat units often show little variability within the framework sequence (i.e. the residue(s) not involved in direct DNA contact (non-RVD residues). This lack of variability may be due to a number of factors including evolutionary relationships between individual TALE repeat units and protein folding requirements between adjacent repeats. Between differing phytopathogenicbacterial species however the framework sequences can vary.
  • the TAL-effector repeat sequences in the Xanthomonas campestris pv vesicatoria, the protein AvrBs3 has less than 40% homology with brgl 1 and hpxl 7 repeat units from Ralstonia solanacearum (see Heuer et al (2007) Appl Environ Micro 73 (13): 4379-4384).
  • the TAL-effector repeat may be under stringent functional selection in each bacterium's natural environment, e.g., from the sequence of the genes in the host plant that the TAL- effector regulates.
  • Variants in the TAL-effector framework can be introduced by targeted or random mutagenesis by various methods known in the art, and the resultant TAL-effector fusion proteins screened for optimal activity.
  • TAL-effector DNA binding domains and/or zinc finger domains may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length (e.g., TGEKP, TGGQRP, TGQKP, and/or TGSQKP), although it is likely that sequences that can function as flanking sequences N-terminal and C-terminal of the TAL domain) would be required at the interface between the TAL-effector repeat domain and the linker.
  • linkers when linkers are used, linkers of five or more amino acids can be used in conjunction with the flanking sequences to join the TAL-effector DNA binding domains to a desired fusion partner domain. See, also, U.S. Patent Nos.
  • linkers between the TAL-effector repeat domains and the fused functional protein domains can be constructed to be either flexible or positionally constrained to allow for the most efficient genomic modification. Linkers of varying lengths and compositions may be tested.
  • the present chimeric peptides may be made using well-established peptide synthetic techniques, for example, FMOC and t-BOC methodologies.
  • polynucleotides disclosed herein, for example, DNA substrates and DNA encoding the present chimeric endonucleases may also be made based on the known sequence information using well-established techniques.
  • Peptides and oligonucleotides are also commercially available.
  • Recombinant technology may also be used to prepare the chimeric endonuclease.
  • a DNA construct comprising DNA encoding the selected nuclease, linking domain (if present), DNA-targeting domain, and any functional domains if present may be inserted into a suitable expression vector which is subsequently introduced into an appropriate host cell (such as bacterial, yeast, algal, fungal, insect, plant and mammalian) for expression.
  • an appropriate host cell such as bacterial, yeast, algal, fungal, insect, plant and mammalian
  • Such transformed host cells are herein characterized as having the chimeric endonuclease DNA incorporated "expressibly" therein.
  • Suitable expression vectors are those vectors which will drive expression of the inserted DNA in the selected host.
  • expression vectors are prepared by site-directed insertion of a DNA construct therein.
  • the DNA construct is prepared by replacing a coding region, or a portion thereof, within a gene native to the selected host, or in a gene originating from a virus infectious to the host, with the endonuclease construct.
  • regions required to control expression of the endonuclease DNA which are recognized by the host, including a promoter and a 3' region to terminate expression, are inherent in the DNA construct.
  • a selection marker is generally included in the vector which takes the form of a gene conferring some survival advantage on the transformants such as antibiotic resistance.
  • Cells stably transformed with endonuclease DNA-containing vector are grown in culture media and under growth conditions that facilitate the growth of the particular host cell used.
  • One of skill in the art would be familiar with the media and other growth conditions.
  • Chimeric endonucleases of the invention comprising a TAL domain may be constructed using techniques well known in the art. One suitable protocol is found in Sanjana Nature Protocols 7: 171-192 (2012) which is specifically incorporated herein by reference.
  • nucleic acid encoding each desired repeat unit may be amplified with ligation adapters that uniquely specify the position of the repeat unit in the TAL domain to create a library that can be reused.
  • Appropriate amplification products may be ligated together into hexamers and then amplified by PCR.
  • the hexamers may be assembled into a suitably prepared plasmid background, for example, using a Golden Gate digestion-ligation.
  • the plasmid backbone may contain a negative selection gene, for example, ccdB, which selects against empty plasmid.
  • the plasmid may be constructed to contain coding sequence for one or more flanking sequences such that insertion of the coding sequence for the TAL domain will be in frame with the flanking sequences resulting in TAL domain comprising flanking sequences.
  • the TAL domain coding sequences, optionally with flanking sequences, can then be combined with the nuclease coding sequences and any other desired coding sequences, for example, nuclear localization sequences (NLS), using standard techniques.
  • NLS nuclear localization sequences
  • the utility of a chimeric endonuclease in accordance with the invention may be confirmed using a DNA substrate designed for the endonuclease.
  • the DNA substrate will include suitable counterpart regions to the nuclease, linking and DNA-targeting domains of the endonuclease.
  • the substrate will include a cleavage motif of the nuclease domain, a DNA spacer that correlates with the linking domain and a binding site for the DNA-targeting domain.
  • a chimeric endonuclease including the I-Tevl nuclease domain at least a portion of the I-Tevl linker as the linking domain and the DNA-targeting domain of a zinc finger (e.g.
  • a suitable substrate will include a cleavage motif of I-Tevl (5 '- CNNNG-3'), a binding site for the selected zinc finger and a DNA spacer that connects the two and which is compatible with the I-Tevl linker to permit interaction between the nuclease and the substrate.
  • the substrate may incorporate a native cleavage motif or may incorporate a cleavage motif derived from the native cleavage motif, i.e. somewhat modified from the native cleavage motif while still recognized and cleaved by the nuclease.
  • the binding site for the DNA- targeting domain may similarly be a native sequence, or may be modified without loss of function.
  • the DNA spacer will be of a size that permits binding of the endonuclease DNA-targeting domain to the substrate binding site, and nuclease access to the cleavage motif.
  • the DNA spacer that links the cleavage motif to the binding site may comprise about 10 to about 30 base pairs, and typically comprises about 15-25 base pairs.
  • the length of the DNA spacer may be adjusted depending on the length of the linker domain and any flanking sequences present in the chimeric endonuclease of the invention.
  • a chimeric endonuclease of the invention is to target a DNA in a ceil
  • the length of the linker may be adjusted such that, upon binding of the DNA-targeting domain to the DNA, the nuclease domain is brought into proximity with the cleavage site.
  • a given DNA substrate is useful in a method of determining the activity of its corresponding chimeric endonuclease.
  • the DNA substrate may be utilitized as pair of complementary olignucleotides annealed together, which may be detectably labeled, e.g. radtoactively labeled.
  • the endonuclease is incubated with its substrate under conditions suitable to permit binding of the endonuclease DNA targeting domain to the binding site on the substrate, and subsequent nuclease cleavage at the cleavage site. Cleavage of the substrate can then be determined using well-established techniques, for example, polyacrylamide gel electrophoresis.
  • the DNA substrate may be incorporated within a vector for use in an assay to determine endonuclease activity.
  • a cell- based bacterial Escherichia coli two-plasmid genetic selection system may be utilized to determine whether or not the chimeric endonuclease can cleave the target cleavage site.
  • the DNA encoding the chimeric endonuclease is incorporated and expressed from one plasm id of the system, and the target DNA substrate is incorporated and expressed from the second plasmid.
  • the target substrate plasmid also encodes a toxin, such as a DNA gyrase toxin.
  • the toxin will not be expressed and the cells, e.g. bacterial cells such as E. coli cells, will survive when plated on selective solid media plates. If the endonuclease cannot cleave the target site, the toxin will be expressed and the cells will not survive on selective media plates.
  • the percentage survival for each combination of fusion and target site is simply the ratio of survival on selective to non-selective plates.
  • a yeast-based assay which utilizes detectable enzyme activity, e.g. beta-galactosidase activity as a readout of endonuclease activity.
  • the lacZ gene is disrupted and partially duplicated in a first plasmid.
  • the DNA substrate is cloned in between the lacZ gene fragments. Cleavage of the substrate by the endonuclease (expressed from a second plasmid) initiates DNA repair and generation of a functional LacZ protein (and beta-gal ctosidase activity).
  • a mammalian cell-based assay which utilizes detectable activity, e.g. the fluorescence of green fluorescent protein (GFP), as a readout of endonuclease activity.
  • detectable activity e.g. the fluorescence of green fluorescent protein (GFP)
  • the GFP gene is disrupted and partially duplicated in a first plasmid.
  • the DNA substrate is cloned in between the GFP gene fragments. Cleavage of the substrate by the endonuclease (expressed from a second plasmid) initiates DNA repair and generation of a functional GFP and fluorescence can be detected.
  • chimeric endonucleases of the invention comprise a nuclease domain that recognizes a 5'CNNNG3' cleavage motif and do not cleave, or cleave at a much reduced level, DNA sequences in which this motif has been altered. See Figure 3e. As shown in Figure 1 1 , the motif is prevalent in human cDNA sequences. Where one allele of a SNP comprises a functional motif and other alleles have a non-functional motif, this difference in reactivity can be used to identify which allele is present in a given sample. This could be useful for high throughput SNP screening for specific disease causing alleles,
  • kits of the invention comprising a chimeric endonuclease and a DNA substrate therefor are provided.
  • kits of the invention may comprise a second plasmid with reporter gene and the DNA binding motif - optimized DNA spacer - and cleavage site.
  • kits of the invention may comprise one or more multicloning sites (MCS) that may be disposed in such a fashion as to permit rapid exchange of nuclease and/or DNA targeting domains.
  • MCS multicloning sites
  • a plasmid may contain MCS-universal linker-MCS
  • kit of the invention may comprise a plasmid encoding an I-TevI-TAL domain chimeric endonuclease.
  • a chimeric endonuclease thus encoded may comprise a linker domain disposed between the nuclease and DNA-targeting domain as well as one or more other functional domains, for example, nuclear localization signals, disposed at either the N-or C-terminal or both.
  • the present chimeric GIY-YIG endonucleases are active in vivo and in vitro, function as monomers, and retain the cleavage specificity associated with the parental GIY-YIG nuclease domain.
  • the G1Y-YTG nuclease domain is shown to be a viable alternative to the Fokl nuclease domain for genome editing applications.
  • a gene includes a DNA region encoding a gene product (which may be a protein or an RNA), as well as all DNA regions which regulate the production of the gene product which may include, but are not limited to, one or more of promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.
  • Methods of the invention typically include introducing one or more chimeric endonucleases and/or nucleic acid molecules encoding such chimeric endonucleases, into one or more cells, which may be isolated or may be part of an organism. Any method of introducing known to those skilled in the art may be used. Examples include direct injection of DNA and/or RNA encoding chimeric endonucleases of the invention, transfection, electroporation, transduction, bombardment, lipofection and the like. Suitable cells include, but are not limited to, eukaryotic and prokaryotic cells. Cells may be cultured cell lines or primary cells.
  • Primary cells will typically be used when it is desired to modify the cell and reintroduce it into the organism from which it was derived.
  • Cells may be from any type of organism, for example, may be mammalian cells, plant cells, algal cells, insect cells, or fungal cells. Suitable types of cell include, but are not limited to, stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, mesenchymal stem cells, muscle stem cells and skin stem cells), In some embodiments, the cells used in the methods of the invention may be plant cells.
  • stem cells e.g., embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, mesenchymal stem cells, muscle stem cells and skin stem cells
  • the cells used in the methods of the invention may be plant cells.
  • DNA constructs encoding chimeric endonucleases of the invention may be introduced into plant cells using Agrobacterium tumefaciens-mediated transformation.
  • Suitable plant cells include, but are not limited to, cells of monocotyledonous (monocots) or dicotyledonous (dicots) plants, plant organs, plant tissues, and seeds.
  • Examples of plant species of interest include, but are not limited to, corn or maize (Zea mays), Brassica sp. (e.g., B.napus, B. rapa, B.
  • j ncea particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum, T Turgidum ssp.
  • millet e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus
  • soybean Glycine max
  • tobacco Nicotiana tabacum
  • potato Solanum tuberosum
  • peanuts Arachis hypogaea
  • cotton Gossypium barbadense, Gossypium hirsutum
  • sweet potato Ipomoea batatus
  • cassava Manihot esculenta
  • coffee Coffea spp.
  • coconut Cocos nucifera
  • pineapple Ananas comosus
  • citrus trees Ciitrus spp.
  • cocoa Theobroma cacao
  • tea Ciamellia sinensis
  • avocado Per sea americana
  • fig Ficus casica
  • guava Psidium guajava
  • mango Manifera indica
  • olive Olea europaea
  • papaya Carica papaya
  • cashew Alpha guapha guajava
  • macadamia Macadamia integrifolia
  • almond Prunus amygdalus
  • sugar beets Beta vulgaris
  • sugarcane sacharum spp.
  • oats barley, vegetables, ornamentals, and conifers.
  • plants for use in methods of the present invention are crop plants (for example, sunflower, Brassica sp., cotton, sugar beet, soybean, peanut, alfalfa, safflower, tobacco, corn, rice, wheat, rye, barley triticale, sorghum, millet, etc.).
  • Plant cells may be from any part of the plant and/or from any stage of plant development.
  • suitable plant cells are those that may be regenerated into plants after being modified using the methods of the invention, for example, cells of a callus.
  • Methods of the invention may also include introducing one or more chimeric endonucleases and/or nucleic acid molecules encoding such chimeric endonucleases, into one or more algal cells. Any species of algae may be used in the methods of the invention.
  • Suitable examples include, but are not limited to, algae of the genus Skeletonema, Thalassiosira, Phaeodactylum, Chaetoceros, Cylindrotheca, Bellerochea, Actinocyclus, Nitzchia, Cyclotella, Isochrysis, Pseudoisochrysis, Dicrateria, Monochrysis, (Pavlova), Tetraselmis (Platymonas), Pyramimonas, Micromonas, Chroomonas, Cryptomonas, Rhodomonas, Chlamydomonas Chlorococcum, OUsthodiscus, Carteria, Dunaliella, or Spirulina.
  • Algal cells may be transformed using the techniques disclosed in United States patent no. 8, 1 19, 859 issued to Vick et al. which is specifically incorporated herein for its teaching of algal transformation.
  • the present invention provides methods of inactivating a gene. Such methods typically comprise introducing a nucleic acid molecule encoding a chimeric endonuclease of the invention into a cell under conditions causing the expression of the chimeric endonuclease.
  • the chimeric endonuclease of the invention can comprise a DNA-targeting domain selected to bind to a gene of interest.
  • the chimeric endonuclease of the invention can cleave the gene of interest leaving a double- stranded break. The normal repair functions in the cell will result in the production of some inserted or deleted bases, which may result in a frame shift thereby inactivating the gene.
  • the chimeric endonuclease may be transiently introduced into the cell. This may be accomplished by transfecting a plasmid with a promoter controlling the expression of the chimeric endonuclease that does not drive expression unless induced, for example, the Tet-On promoter. Alternatively, transient expression may be accomplished by introducing mRNA encoding the chimeric endonuclease of the invention into the cell. Normal housekeeping functions of the cell will degrade the mRNA over time thereby stopping expression of the chimeric endonuclease.
  • Methods of the invention also include methods of changing the nucleic acid sequence of a gene.
  • a nucleic acid molecule encoding a chimeric endonuclease of the invention is introduced into a target cell under conditions causing the expression of the chimeric endonuclease.
  • the chimeric endonuclease of the invention is constructed so as to bind to and cleave a gene of interest.
  • a second nucleic acid molecule comprising a region having a nucleotide sequence that has a high degree of sequence identity to the gene in the region of the cleavage site is introduced into the cell.
  • the region of high sequence identity may have a length of from about 10 basepairs to about 1000 basepairs, from about 25 basepairs to about 1000 basepairs, from about 50 basepairs to about 1000 basepairs, from about 75 basepairs to about 1000 basepairs from about 100 basepairs to about 1000 basepairs, from about 200 basepairs to about 1000 basepairs, from about 300 basepairs to about 1000 basepairs, from about 400 basepairs to about 1000 basepairs, from about 500 basepairs to about 1000 basepairs, from about 750 basepairs to about 1000 basepairs, from about 10 basepairs to about 500 basepairs, from about 25 basepairs to about 500 basepairs, from about 50 basepairs to about 500 basepairs, from about 75 basepairs to about 500 basepairs from about 100 basepairs to about 500 basepairs, from about 200 basepairs to about 500 basepairs, from about 300
  • High sequence identity means the region and the corresponding region in the gene have a sequence identity of from about 80% to about 100%, from about 82% to about 100%, from about 86% to about 100%, from about 88% to about 100%, from about 90% to about 100%, from about 92% to about 100%, from about 94% to about 100%, from about 96% to about 100%, from about 98% to about 100%, or from about 80% to about 95%, from about 82% to about 95%, from about 86% to about 95%, from about 88% to about 95%, from about 90% to about 95%, from about 92% to about 95%, or from about 80% to about 90%, from about 82% to about 90%, from about 86% to about 90%, from about 88% to about 90%.
  • the region may comprise an altered sequence when compared to the gene of interest, for example, may have one or more mutations that will result in changes to one or more amino acids in a protein encoded by the gene.
  • the double-stranded break introduced by the chimeric endonuclease of the invention may be repaired by homologous recombination with the region of high sequence identity of the second nucleic acid, effectively substituting all or a portion of the sequence of the homologous region in the second nucleic acid molecule for the original sequence of the gene. This results in a gene with modified nucleic acid sequence.
  • the chimeric endonuclease of the invention is transiently expressed in the cell.
  • transient expression may be accomplished by introducing mRNA encoding the chimeric endonuclease of the invention into the cell. Normal housekeeping functions of the cell will degrade the mRNA over time thereby stopping expression of the chimeric endonuclease.
  • the second nucleic acid molecule may be a linear DNA molecule.
  • Methods of the invention also include methods of deleting all or a portion of the nucleic acid sequence of a gene.
  • a nucleic acid molecule encoding a chimeric endonuclease of the invention is introduced into a target cell under conditions causing the expression of the chimeric endonuclease.
  • the chimeric endonuclease of the invention is constructed so as to bind to and cleave a gene of interest.
  • a second nucleic acid molecule comprising a region having a nucleotide sequence that has a high degree of sequence identity to the gene in the region of the cleavage site is introduced into the cell.
  • the region of high sequence identity is as described above except that the region will lack sequence corresponding to the portions of the gene adjacent to the anticipated cleavage site. After homologous recombination beween the gene and the second nucleic acid molecule, the lacking sequence will appear as a deletion of the sequence of the gene. Any number of basepairs may be lacking, from 1 to the entire sequence of the gene.
  • the double strand break introduced by the chimeric endonuclease of the invention may be repaired by homologous recombination with the region of high sequence identity of the second nucleic acid, effectively substituting all or a portion of the sequence of the region of high sequence identity for the original sequence of the gene.
  • the chimeric endonuclease of the invention is transiently expressed in the cell. This may be accomplished by transfecting a plasmid with a promoter controlling the expression of the chimeric endonuclease that does not drive expression unless induced, for example, the Tet-On promoter. Alternatively, transient expression may be accomplished by introducing mRNA encoding the chimeric endonuclease of the invention into the cell.
  • Methods of the invention also include methods of making a cell having an altered genome.
  • the altered genome may comprise an inactivated gene.
  • the altered genome may comprise a gene having one or more mutations.
  • the altered genome may lack all or a portion of a gene.
  • a nucleic acid molecule encoding a chimeric endonuclease of the invention is introduced into a target cell under conditions causing the expression of the chimeric endonuclease.
  • the chimeric endonuclease of the invention is constructed so as to bind to and cleave a gene of interest. Cleavage of the target and DNA repair will result in an inactivated gene.
  • a nucleic acid molecule encoding a chimeric endonuclease of the invention is introduced into a target cell under conditions causing the expression of the chimeric endonuclease.
  • a second nucleic acid molecule comprising a region having a nucleotide sequence that has a high degree of sequence identity to the gene in the region of the cleavage site is introduced into the cell. The region is as described above.
  • the region may comprise an altered sequence when compared to the gene of interest, for example, may have one or more mutations that will result in changes to one or more amino acids in a protein encoded by the gene.
  • the double-stranded break introduced by the chimeric endonuclease of the invention may be repaired by homologous recombination with the region of high sequence identity of the second nucleic acid, effectively substituting all or a portion of the sequence of the region of high sequence homology in the second nucleic acid molecule for the original sequence of the gene. This results in a cell with an altered genome.
  • a nucleic acid molecule encoding a chimeric endonuclease of the invention is introduced into a target cell under conditions causing the expression of the chimeric endonuclease.
  • the chimeric endonuclease of the invention is constructed so as to bind to and cleave a gene of interest.
  • a second nucleic acid molecule comprising a region having a nucleotide sequence that has a high degree of sequence identity to the gene in the region of the cleavage site is introduced into the cell. The region typically lacks the sequence of the gene adjacent to the cleavage site, i.e.
  • the chimeric endonuclease of the invention has a deletion that encompasses the anticipated cleavage site.
  • the double-stranded break introduced by the chimeric endonuclease of the invention may be repaired by homologous recombination with the region of high sequence identity of the second nucleic acid, effectively substituting all or a portion of the sequence of the region for the original sequence of the gene. Since this region contains a deletion at the cleavage site of the chimeric endonuclease of the invention, this results in a gene with a deletion in its nucleic acid sequence.
  • the chimeric endonuclease of the invention is transiently expressed in the cell.
  • transient expression may be accomplished by introducing mRNA encoding the chimeric endonuclease of the invention into the cell. Normal housekeeping functions of the cell will degrade the mRNA over time thereby stopping expression of the chimeric endonuclease.
  • the second nucleic acid molecule may be a linear DNA molecule.
  • the present invention includes cells produced using the methods of the invention.
  • Cells of the invention will typically comprise one or more alterations in their genome.
  • “genome” encompasses the genetic material present in cellular compartments, such as chloroplasts, mitochondria and the like, as well as genetic material in the nucleus of the cell.
  • the present invention encompasses cells of any type, for example, cultured cells, which may be from an established cell line or may be a primary culture, cells from single cell organisms and cells from multicellular organisms. Cells from multicellular organisms may be isolated as individual cells, present in an organ, which may be isolated, or present in the whole organism.
  • the cells may be prokaryotic or eukaryotic. Any type of mammalian cells, for example mice, rat, primate (especially human primate), chicken, porcine, bovine, equine cells, may be used. Either primary cultured cells or an established cell line can be employed.
  • the primary cultured cells may originate from any tissue, e.g. cartilage, bone, skin, nerve, oral alimentary canal, liver, pancreas, kidney, gland, heart, muscle, tendon, fat, connective, reproductive organ tissue, ocular, blood vessel, bone marrow and blood.
  • Exemplary cell types include osteoblasts, keratinocytes, melanocytes, hepatocytes, gliacytes, pancreatic beta cells, pancreatic exocrine cells, neural stem cells, neural precursor cells, spinal cord precursor cells, nerve cells, mammary gland cells, salivary gland cells, renal glomerular endothelial cells, tubular epithelial cells, adrenocortical and adrenomedullary cells, cardiomyocytes, chondrocytes, skeletal and smooth muscle cells, fat and fat precursor cells, corneal and crystalline lens cells, embryonic retina cells, vascular cells, endothelial cells, bone marrow stromal cells and lymphocytes.
  • the methods of the invention may be employed to create muscle cells (smooth, skeletal, cardiac), connective tissue cells (fibroblasts, monocytes, mast cells, granulocytes, plasma cells, osteoclasts, osteoblasts, osteocytes, chondrocytes), epithelial cells (from skin, gastrointestinal, urinary tract or reproductive tract, or organ epithelial cells from the liver, pancreas or spleen), or nervous system cells (glial, neuronal, astrocytes), wherein the cells have an altered genome relative to the cells that were used as starting material in the methods of the invention.
  • muscle cells smooth, skeletal, cardiac
  • connective tissue cells fibroblasts, monocytes, mast cells, granulocytes, plasma cells, osteoclasts, osteoblasts, osteocytes, chondrocytes
  • epithelial cells from skin, gastrointestinal, urinary tract or reproductive tract, or organ epithelial cells from the liver, pancreas or spleen
  • nervous system cells
  • mammalian stem cells may be used in the practice of the invention.
  • exemplary stem cells include, but are not limited to, ectodermal, mesodermal, endodermal, mesenchymal, hematopoietic, neural, hepatic, muscle, pancreatic, cutaneous, retinal and follicular stem cells.
  • non-mammalian cells from any non- mammalian organism may also be used in the practice of the invention.
  • DSMZ the German National Resource Centre for Biological Material is one
  • ATCC the American Type Culture Collection is another.
  • Cells from any of the known repositories may be advantageously used in the practice of the invention.
  • cells of the invention may be algal cells comprising one or more alterations in their genome relative to corresponding wildtype algal cell.
  • Chimeric endonucleases of the invention may be used for in biological research by providing a mechanism to manipulate the genome of a cell or organism. Such genome editing allows the elucidation of the role of individual genes and portions of genes by allowing the controlled introduction of changes into the genome. This will allow the production of customized cells that are suitable for use in screening.
  • the present invention also permits gene therapy, for example, by correcting a genetic defect using the materials and methods described herein.
  • the present methods are particularly well suited for ex vivo methods of gene therapy where cells are removed from a patient, manipulated to achieve a desired outcome, and reintroduced in the patient.
  • Materials and methods of the invention will find use in agricultural for creation of plants having improved growth rate , tolerance to stresses such as drought and pests, and taste. Materials and methods of the invention will find application in molecular biology and diagnostics by allowing the direct manipulation of any desired target DNA.
  • Escherichia coli strains DH5a and ER2566 were used for plasmid manipulations and protein expression, respectively.
  • the ryA zinc-finger gene was synthesized by Integrated DNA
  • the R27A mutants of Tev-ZFEs were generated using Quickchange mutagenesis (DE613/614). The sequences of all GIY-ZFEs constructed are listed in Fig. 4).
  • the hybrid target sites (Fig. IB and 2Q were cloned into the toxic reporter plasmid p i 1 -lacY-wtxl to generate pToxTZ1.35 and pToxBZl ,35.
  • Identical Tev-ryA and Bmo-ryA target sites were generated in pSP72 for in vitro cleavage assays.
  • the Tev-ryA site hybrid homing site was also cloned into LITMUS28i using BamHI and Xhol to generate pTZHS1.35.
  • the two-site Tev-ZF plasmids were created by sub-cloning the PvuII/Hpal fragment from pSP-TZHS1.35 into the Swal site of pTZHS 1.35 to generate pTZHS2.35 and pTZHS3.35 (with the second TZHS in either orientation).
  • the G5A or C I A/G5A mutations were introduced into pToxTZ and pTZHS plasmids by Quickchange mutagenesis. All constructs were verified by sequencing.
  • the two plasmid genetic selection was performed as described with toxic (reporter) plasmids containing hybrid Tev- or Bmo-ryA target sites, or mutant ryA target sites (with G5A or C1A/G5A substitutions), or plasmids lacking a target site (pi 1-lacY-wtxl). Survival percentage was calculated by dividing the number of colonies observed on selective by those observed on non-selective plates.
  • the cell lysate was clarified by centriiugation at 20400 x g, followed by sonication for 30 seconds, and centriiugation at 20400 x g for 15 minutes.
  • the clarified lysate was loaded onto a 1 mL HisTrap-HP column (GE Healthcare), washed with 15 mL binding buffer and then 10 mL wash buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 50 mM imidazole, 5% glycerol, and 1 mM DDT). Bound proteins were eluted in 1.5 mL fractions in four 5 mL step elutions with increasing concentrations of imidazole.
  • Tev-ZFE were performed in buffer containing 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM MgCl 2 , 5% glycerol, I mM DTT and 10 nM pTZHS1.33. Reactions were incubated for 3 minutes at 37°C, stopped with 5 ⁇ stop solution (100 mM EDTA, 40% glycerol, and bromophenol blue), and electrophoresed on a 1% agarose gel prior to staining with ethiduium bromide and analysis on an AlphalmagerTM3400 (Alpha Innotech). The EC 0 5ma x was determined by fitting the data to the equation
  • ⁇ ([endoj) is the fraction of substrate cleaved at concentration of TevN201 -ZFE [endo]
  • /nax is the maximal fraction cleavage, with 1 being the highest value
  • H is the Hill constant that was set to 1.
  • the initial reaction velocity was determined using supercoiled plasmid substrate with varying concentrations of TevN201 -ZFE (0.7 nM to 47 nM) and buffer as above. Aliquots were removed at various times, stopped and analyzed as above. The data for product appearance was fitted to the equation
  • [C] [Co]expHt, where [C] is the concentration (nM) of supercoiled plasmid at time t, [C 0 ] is the initial concentration of supercoiled substrate (nM), and k ⁇ is the first order rate constant (in
  • GIY-YIG homing endonucleases function as monomers
  • GIY-ZFEs GlY-YIG-zinc finger endonucleases
  • the DNA substrates consisted of 31 to 33 bps of the I-TevI td homing site that is contacted by the linker and nuclease domains, joined to the 9-bp ryA target site (Fig. IB).
  • the critical G of the 5'-CXXXG-3' cleavage motif is positioned 28-bp distant from the ryA binding site, in analogy with the native spacing of the I-TevI td homing site.
  • An analogous set of I-BmoI-ryA fusions were constructed (Bmo-ZFEs, Fig. 2C ).
  • GIY-ZFEs require specific sequences for efficient cleavage
  • Both I-Tevl and I-Bmol are DNA endonucleases that cleave specific sequences at a defined distance from their primary binding sites.
  • the TevN201-ZFE and BmoN221 -ZFE fusions were purified for in vitro mapping studies (Fig. >A and 3B).
  • Fig. >A and 3B Using strand-specific end-labeled substrates, the bottom- and top- strand nicking sites of TevN201-ZFE were mapped to lie within the 5'-CXXXG-3' motif, with ⁇ and J, representing the bottom- and top-strand nicking sites, respectively (Fig. 3Q.
  • GIY-ZF s function as monomers
  • cleavage assays were performed to determine the relationship between TevN201-ZFE enzyme concentration and initial reaction velocity.
  • the reaction progress curves indicated an initial burst of cleavage followed by a slower rate of product accumulation (Fig. 5A), consistent with product release being the rate-limiting step.
  • the initial burst phase was used to estimate initial velocity, and plotting against protein concentration yielded a linear relationship (Fig. 5A), suggesting that DNA hydrolysis catalyzed by TevN201 -ZFE is first order with respect to protein concentration.
  • Time-course cleavage assays under single-turnover conditions were also conducted with plasmids that contained one or two Tev-ryA target sites. Two-site plasmids that differed in whether the target sites were in the same or opposite orientations relative to each other were constructed.
  • the substrates consisted of various lengths of the native I-Tevl target sequence derived from the phage T4 td gene that were fused to the 5' end of the PthXol TAL-effector binding site.
  • the substrates are designated TP (for Tev- PthXol), and number according the length of the I-Tevl target site included (TP24 has 24 bp of the I-Tev] target site).
  • the substrates were designed as complementary oligonucleotides that were subsequently annealed and cloned into pLitmus. Alternatively, the oligonucleotides were radiolabeled with 32 P, and then annealed.
  • the radioactively labeled DNA substrates were used to map the cleavage sites of the Tev-TAL fusions.
  • the substrates were labeled on both strands, meaning that both the top and bottom strand cleavage products could be mapped.
  • two prominent cleavage products were observed with the TP series of substrate when incubated with Tev201-TAL.
  • the size of the bottom strand product varies with the TP substrate tested. The size difference is due to the fact that the position of the bottom strand cleavage site is moved closer to the 3' end of the duplex DNA substrate (i.e. closer to the TAL binding site) because the shorter TP substrates include less of the native I-Tevl site.
  • the top strand cleavage site does not change size, because its position relative to the 5' end of the duplex substrates does not change in any of the substrates.
  • the sizes of both cleavage products are consistent with specific cleavage by the Tev201 -TAL fusion at the CNNNG cleavage motif.
  • I-TevI, and 1-BmoI indicate the regions of conservation and consensus. Indicated is the functionally critical region of the ITevI linker (Kowalski et al. 1999 NAR; Liu et al. 2008, JMB).
  • an optimized linker may be generated that includes deletion, replacement, and addition of amino acid sequences using conventional methods. This may include the replacement of the functionally non-critical regions in the linker with other desired sequences.
  • the nucleotide requirements of the I-TevI linker (residues 97-169) for its corresponding region on a substrate was determined.
  • a coupled in vitro/in vivo selection system was used (Edgell et al. Current Biology (2003) 13:973-978) that relies on cleavage of a randomized DNA spacer plasmid library by the Tev l 69-Onu fusion protein (see Fig. 18 for amino acid sequences of a family of Tev-Onu fusion products that vary in the size of the Tev portion).
  • Cleaved substrates are isolated, and amplified in E. coli, followed by bar-coded PCR for deep-sequencing on an Ion Torrent sequencer.
  • the I-TevI linker has a nucleotide preference at 3 positions within the DNA spacer, namely, positions 2, 8 and 15 (see Figure l Oa b).
  • a consensus DNA sequence for the Tevl 69 constructs could be 5' CNNNGN(A/T)NNNNNG(A/T), where N is any nucleotide and the CNNNG is the required cleavage motif. This motif occurs in >93% of all non-redundant human cDNAs at least once (see Figure 1 1 ).
  • Figure I 0c demonstrates the relationship between the nucleotide bias in the DNA spacer region (bottom), and its relationship to the evolutionary conserved amino acids of the I-Tevl native target gene thymidylate synthase in bacteriophage T4 (spp). Domain knowledge regarding the original sequence permits refinement of the spacer region identified in Figure 10b to identify potential artifacts linked to the original sequence bias to generate a viable consensus and indicates the importance of the core spacer sequence comprising CNNGN(AZT), and the scaled optional nature of an additional NNN G and the additional terminal (A T) nucleotide.
  • Tev-TAL fusions were constructed using standard molecular biology cloning techniques by fusing different lengths of the I-Tevl nuclease domain to different N-terminal residues of the TAL effector PthXol .
  • the general schematic of the fusions is shown in Figure 22, and the constructs that have been rigorously tested are shown in Table 4. Note that the longest TAL domain used corresponds to essentially the full length TAL effector protein. TAL domains that are truncated at the N-terminal side are labeled by the amino acid residues used as the fusion point (ie. T120 is threonine 120). Model DNA substrates were also constructed to test activity of the Tev-TAL fusions.
  • the substrates are shown in the 5'-3' direction in Figure 22.
  • the substrates are tri-partite, and consist of the I-Tevl cleavage motif (CAACG), a variable length spacer, and a TAL effector-binding site.
  • CAACG I-Tevl cleavage motif
  • the Tev-TAL target site is cloned on a plasmid between an interrupted and partially duplicated lacZ coding for a nonfunctional beta-galactosidase enzyme.
  • a separate plasmid expresses the Tev-TAL fusion under the control of either a weak or strong constitutive promoter.
  • Separate yeast strains harboring each plasmid are mated, and a functional lacZ gene (and beta- galactosidase activity) is only produced when the Tev-TAL fusion cleaves its target, promoting DNA repair of the lacZ gene to generate a functional copy.
  • FIG. 23 Shown in Figure 23 are representative data for a set of the Tev-TAL fusions tested in the yeast-based assay against substrates differing in the spacer length.
  • Our data indicate that the N-terminal fusion point on the TAL effector has a major influence on the activity of the Tev-TAL fusions.
  • the data also show that the fusions consisting of the longer I-Tevl fragments (residues 1-201 or 1 -206) are most active on the DNA substrates with the longest spacer lengths, consistent with the I- TevI linker region acting as a ruler to position the nuclease domain at the CNNNG cleavage motif.
  • the experiments shown in Figure 23 addressed the length requirement of the DNA spacer portion of the I-Tevl linker.
  • the optimal DNA spacer length is 15 bp.
  • the DNA spacer is the region of substrate that is contacted by the I- TevI linker, which may require specific nucleotides for contact.
  • the native I-Tevl target lies within the phage T4 thymidylate synthase gene, and we rationalized that testing activity on a series of related thymidylate synthase genes from other phage would inform us of nucleotide requirements in the DNA spacer.
  • the Tev-TAL12 fusion showed activity on par with or better than the native T4 sequence (TP1.15).
  • the Tev-TAL12 fusion was ⁇ 1.5-fold more active on the substrate derived from Tula phage than on its native substrate.
  • Other phage-derived thymidylate synthase substrates exhibited a range of activity.
  • all the substrates had multiple substitutions in the DNA spacer relative to the T4 sequence (indicated by lower case red text), indicating that the I-Tevl linker can tolerate multiple substitutions in the DNA spacer.
  • the only exception was the substrate derived from the RB32 phage, notable because this substrate contains mutations that are also found in the other substrates tested.
  • the RB32-derived substrate had a C to A mutation in the cleavage motif relative to the other substrates, which may explain the lower activity of the Tev- TAL12 fusion.
  • Targeted manipulation of complex genomes is greatly enhanced by site-specific DNA endonucleases that can introduce a nick or double-strand break at specific locations within a genome.
  • DNA endonucleases derived from naturally occurring homing endonucleases.
  • Fokl the dimeric non-specific nuclease domain derived from the type IIS restriction enzyme Fokl that is fused to the C-terminaus of zinc-fingers or TAL effector domains, to create zinc-finger nucleases (ZFNs) or TAL effector nucleases (TALENs).
  • Tev-TAL fusions represent a third technology for genome engineering applications including, but not limited to, targeted cleavage of a clinically relevant sequence in the human genome or genome of model organisms (mouse, rat, Drosophila, etc) for gene therapy purposes, or targeted cleavage of sequences in genomes to introduce mutagenic lesions with the goal of creating gene knockouts.
  • the GIY-YIG nuclease domains used in the present invention do not impose the same design limitations.
  • the GIY-YIG nuclease domains of the present invention will be useful for introducing targeted double-strand breaks at sequences defined by a distinct DNA- targeting domain.
  • the applications of the GIY-YIG nuclease fusions is an improvement over ZFNs, TALENS, or engineered homing endonucleases, notably for targeted manipulation of complex genomes for gene replacement, or gene knockouts.
  • Tev-TAL fusions of the present invention are different from existing nucleases that fuse the Fokl nuclease domain to the C-terminus of the TAL effector domain (the TALENs) in at least:
  • the Tev-TAL nuclease needs a single DNA target site for cleavage where as the TALENs need two DNA target sites (the Tev-TALs function as monomers, whereas TALENs function as dimers). This reduces the engineering requirements by a factor of 2, and also reduces the complexity of finding two suitable target sites in close proximity (as needed with ZFNs and TALENs);
  • Tev-TAL fusions use different lengths of the I-Tevl nuclease domain and PthXol TAL effector.
  • the nuclease domain from the restriction enzyme PvuII has been fused to zinc-fingers and to a catalytically inactive LAGLIDADG homing endonuclease to create novel site-specific nucleases.
  • the Puvll nuclease functions as a dimer, like Fokl, and has a 6-bp cleavage site requirement (in addition to the targeting requirements of the zinc finger or LAGLIDADG homing endonuclease).
  • Example 5 Bacteria and yeast strains. Escherichia coli strains DH5a and ER2566
  • E. coli strains were grown in Luria-Broth media supplemented with the appropriate antibiotics.
  • Sachoramyces cerevisiae strains YPH500(a) and YPH499(a) were used for the single-strand annealing assay, and grown in appropriate media as described (CHRISTIAN et al. 2010 Genetics 186: 757-761).
  • mTALENs and substrate plasm ids were constructed by first cloning oligonucleotides corresponding to the target site into the Bglll/Sphl sites of pTox. Each substrate, differing in the DNA spacer length, was PCR amplified with flanking primers and cloned into the Bglll/Sphl sites of the yeast vector pCP5.1 to create the TP series of plasmids (TPS- TPS ⁇ for the yeast activity assay. Mammalian substrates were constructed in the same manner, and cloned into the Sacl/Xhol sites of pcDNA3(+).
  • mTALENs were first constructed in pACYC by changing the Ncol site to Pcil, and by inserting a stop codon downstream of the Bglll site, and the full-length PthXol TAL effector was then cloned into the BamHI/Bglll sites. The I-TevI nuclease domain and various linker lengths were then cloned into the Pcil/BamHI sites. mTALENs that differed in the N-terminal fusion point were constructed by first removing the N-terminal BamHI/Sphl fragment from PthXol , leaving the RVD repeats intact.
  • PCR products corresponding to the new N-terminal fusion point were then cloned into the BamHI/Sphl sites, and the I-TevI nuclease domain was cloned Pcil BamHI.
  • each mTALEN construct digested with Pcil Xhol and subcloned into the Ncol Sall sites of pGPD423 (ALBERTI et al. 2007 Yeast 24: 913-919.).
  • the pACYC backbone was first modified by including an RsrII site upstream of the Pcil site, and mTALEN constructs cloned as above. mTALENs were subsequently cloned into the Pstl RsrII sites of pExodus. A list of mTALENs tested for activity is found in the following table.
  • Fractions containing 250- 325 mM NaCl were pooled, dialyzed, and applied to a SP-FF column (GE Healthcare) equilibrated in buffer A, and eluted in steps of 200 nM NaCl to a final concentration 1 M NaCl.
  • the 400 mM elutions were pooled, and applied to a FF-Q column (GE Healthcare) equilibrated in buffer A, and eluted in steps of 200 mM NaCl.
  • the 400 mM fractions were pooled, concentrated to 0.5 mis and loaded onto a 30-ml Superose 12 gel filtration column (GE Healthcare) equilibrated in buffer A, and 0.25 ml factions collected over 1 column volume.
  • Endonuclease assays on substrates with different length spacers utilized oligonucleotides end-labeled at the 5 ' end with T4 polynucleotide kinase and 32 ⁇ - ⁇ prior to annealing.
  • Cleavage reactions consisted of 20- ⁇ 1 reactions in 1 X NEBuffer 3 reaction buffer for 10 mins at 37°C, and were resolved on 10% denaturing urea-polyarcylamide gels. Mapping of cleavage sites utilized supercoiled pSP72-TP15 in 20- ⁇ volumes of 1 X NEBuffer 3 and a 5-fold molar excess of protein to DNA. Linear cleavage products were gel isolated and set for sequencing at the London Regional Genomics Facility. Cleavage sites were determined from ABI traces, taking into account the additional A added by Taq polymerase during sequencing reactions.
  • yeast reporter assay was performed as described (CHRISTIAN et al. 2010). The protocol was adapted to microtitre plates, where three transformants of YPH499 harbouring the target plasmids (in pCP5.1 ) and YPH500 harbouring the mTALENs were grown in 96-well plates at 30°C overnight with shaking in synthetic complete medium lacking tryptophan and uracil (for the YPH499 target strain) or histidine (for the YPH500 mTALENs strains).
  • the mTALEN and target strains were mated by combining 200 - 500 ⁇ of overnight cultures and adding 1 ml of YPD media, and incubated for 4-6 hrs without shaking at 30°C. Cell density was measured 595 nM by plate reader. Cells were harvested by centrifugation, resuspended and lysed using YeastBuster Protein Extraction Reagent (Novagen) according to the manufacturer's protocol. A total of 60 ⁇ of lysate was transferred to a 96-well plate and ⁇ -galactosidase activity measured and normalized as previously described (TOWNSEND et al. 2009 Nature 459: 442-445.).
  • Miller units were normalized to a SurB dimeric Fokl-TALEN or Zif268 zinc finger nuclease control for assays profiling the optimal mTALEN DNA spacer length, or to the N169-T120 mTALEN on the TP15 substrate for assays profiling CNNNG cleavage site and DNA spacer requirements.
  • HEK 293T cells obtained from ATCC were cultured in high glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), at 37°C in 5% C0 2 . Approximately 2.5xl0 6 million cells were seeded 24 hrs prior to transfection in 6 cm plates. Cells were co-trans fected with 3 g of pExodus mTALEN and 3 ⁇ g of pcDNA3(+) TP15 target DNA using calcium phosphate, and incubated at 37°C with 5% C0 2 for 16 hrs before replacing media.
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • Plasmid DNA was isolated using the BioBasic miniprep kit. Target sites were PCR amplified and gel purified. After gel purification, 250 ng of each PCR producted was incubated with 2U of Ddel (N.E.B.) in 1 X NEBuffer 2 for 1 hr at 37°C. Digests were electrophoresed on a 1 .5% agarose gel and stained with ethidium bromide before analysis on an AlphalmagerTM3400 (Alpha Innotech).
  • PBS phosphate buffered saline
  • Tev- PthXol mTALEN constructs are named using the length of 1-Tevl fragment, beginning at residue 1, followed by the N-terminal residue in PthXo l .
  • All Tev- PthXol mTALENs were tested against model DNA substrates derived from the phage T4 td gene fused to the perfect match PthXol binding site ( Figure 22B).
  • the TevPth (TP) substrates mimic the modularity and orientation of the mTALENs as they consist of, in the 5' to 3 ' direction, a CNNNG cleavage motif, a DNA spacer (normally contacted by the I-Tevl linker), and the PthXol binding site.
  • S206 fragment of I-Tevl contains the entire region of the native I-Tevl linker, including all residues that are known to make base-specific contacts to substrate in the context of the native enzyme (VAN ROEY et al. 2001 EMBO J 20: 3631 -3637).
  • VAN ROEY et al. 2001 EMBO J 20: 3631 -3637 residues that are known to make base-specific contacts to substrate in the context of the native enzyme
  • I-Tevl fragments consisting of residues 1 -169 (N169) and 1-184 (D184) displayed high activity in the context of the T120 or V I 52 PthXol N-terminal fusion points, and both of these fusions also exhibited a 10-bp periodic activity on substrates with varying length spacers (Figure 26B).
  • the top-strand product is a constant length as the 5' end is always the same distance from the 5'-CNNNG-3 ' cleavage motif regardless of the DNA spacer length.
  • the bottom-strand product's size varies proportionally with the distance of the 5'-CNNNG-3' cleavage motif to the TALE binding site.
  • the CS(-) substrates place a CNNNG motif 10-bp from the TALE site, which our spacer length data show is non-permissive for cleavage, consistent with the I-Tevl linker functioning as a molecular rule to position the cleavage domain on substrate.
  • these data show that the I-Tevl catalytic domain has a preferred 5'-CNNNG-3' cleavage motif on native substrate, and that inappropriately spaced, secondary motifs do not support cleavage.
  • TevI component of mTALENs consists of both the I-Tevl nuclease domain (residues 1 -92) and varying lengths of the I-Tevl linker that presumably contact the DNA spacer region of substrate.
  • the linker accurately positions the nuclease domain on the substrate to cleave at the 5'-CAACG-3' motif (DEAN et al. 2002 Proc Natl Acad Sci U S A 99: 8554-8561.).
  • Previous in vitro cleavage assays on partially randomized substrates revealed that wild-type I-TevI can accommodate nucleotide substitutions in the DNA spacer (BRYK et al 1 93), yet it is unknown if the I-TevI linker can tolerate nucleotide substitutions in the context of engineered DNA-binding domains.
  • the TP 15N region was sequenced from 49 active and 62 inactive clones, and the average identity for both sets of clones to the TP15 wild-type sequence was 27%.
  • a positive value for a particular nucleotide indicates enrichment in the active clones, while a negative value indicates selection against that nucleotide. Selection was most evident at three positions (1, 2, and 7), paralleling the activity of some single nucleotide substitutions at these positions ( Figure 29A), while little preference was observed at the remaining positions.
  • G at position 1 may reflect selection of an alternative G to position the nuclease domain to nick the top-strand.
  • nucleotide preferences in Figure 3 OB are generated from relatively small number of sequences, they show that the I- TevI linker domain is extremely tolerant of multiple substitutions within the DNA spacer, as supported by cleavage of td sequences derived from a variety of phage ( Figure 29B).
  • sequence-dependent effects play a significant role in modulating cleavage activity, largely mitigating the effect of any single nucleotide substitution.
  • mTALEN activity was determined by co-transfecting the N169-T120 and D184-V152 constructs with an episomal substrate plasmid containing the hybrid ld/Pt Xo ⁇ target with a 15-bp DNA spacer separating the CAACG motif and TALE binding site ( Figure 31C).
  • the substrate contains a Ddel site immediately adjacent to the I-Tevl cleavage site, allowing us to estimate mTALEN cleavage efficiency as the proportion of subsequently PCR-amplified substrates that were rendered resistant to Ddel digestion as a result of mTALEN cleavage and non homologous end joining (NHEJ) mediated mutagenic repair (Figure 31C).
  • NHEJ non homologous end joining
  • the mTALENs function in HE 293T cells at a level sufficient to induce mutagenic DNA repair on an episomal substrate, and this level of activity is comparable to many reported Fokl-TALENs.
  • mTALENs are a viable alternative genome-editing tool.
  • One obvious advantage of the mTALEN platform is the monomeric nature, simplifying design requirements to target desired sequences.
  • the moderate sequence requirements of the nuclease domain retained in mTALENs can be exploited to minimize off-targeting, or possibly to simplify constructs further by minimizing the number of TALE repeats incorporated.

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Abstract

L'invention concerne une endonucléase chimère qui comprend le domaine de nucléase GIY-YIG qui est lié à un domaine de ciblage d'ADN par un domaine de liaison. Cette endonucléase est utile dans l'édition génomique.
PCT/US2014/014491 2013-02-01 2014-02-03 Endonucléase pour édition génomique Ceased WO2014121222A1 (fr)

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EP2758537A4 (fr) * 2011-09-23 2015-08-12 Univ Iowa State Res Found Architecture de monomère de nucléase tal ou de nucléase à doigt de zinc pour modification d'adn
WO2020225719A1 (fr) 2019-05-03 2020-11-12 Specific Biologics Inc. Endonucléase à double clivage encapsulée dans des lipides pour adn et gène
WO2020260899A1 (fr) 2019-06-27 2020-12-30 Azeria Therapeutics Limited Criblage d'inhibiteurs
WO2021119563A1 (fr) * 2019-12-13 2021-06-17 Inscripta, Inc. Nouvelles enzymes
WO2022097070A1 (fr) * 2020-11-04 2022-05-12 Specific Biologics Inc. Édition de gènes avec une endonucléase modifiée
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WO2011064751A1 (fr) * 2009-11-27 2011-06-03 Basf Plant Science Company Gmbh Endonucléases chimériques et utilisations de celles-ci
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2758537A4 (fr) * 2011-09-23 2015-08-12 Univ Iowa State Res Found Architecture de monomère de nucléase tal ou de nucléase à doigt de zinc pour modification d'adn
GB2600568B (en) * 2019-05-03 2024-07-31 Specific Biologics Inc Lipid-encapsulated dual-cleaving endonuclease for DNA and gene editing
GB2600568A (en) * 2019-05-03 2022-05-04 Specific Biologics Inc Lipid-encapsulated dual-cleaving endonuclease for DNA and gene editing
CN114761550A (zh) * 2019-05-03 2022-07-15 特定生物制品公司 用于dna和基因编辑的脂质包封的双切割内切核酸酶
US11814658B2 (en) 2019-05-03 2023-11-14 Specific Biologics Inc. Lipid-encapsulated dual-cleaving endonuclease for DNA and gene editing
WO2020225719A1 (fr) 2019-05-03 2020-11-12 Specific Biologics Inc. Endonucléase à double clivage encapsulée dans des lipides pour adn et gène
US12297467B2 (en) 2019-05-03 2025-05-13 Specific Biologics Inc. Lipid-encapsulated dual-cleaving endonuclease for DNA and gene editing
US12312615B2 (en) 2019-05-03 2025-05-27 Specific Biologics Inc. Lipid-encapsulated dual-cleaving endonuclease for DNA and gene editing
US12460192B2 (en) 2019-05-03 2025-11-04 Specific Biologics Inc. Lipid-encapsulated dual-cleaving endonuclease for DNA and gene editing
WO2020260899A1 (fr) 2019-06-27 2020-12-30 Azeria Therapeutics Limited Criblage d'inhibiteurs
WO2021119563A1 (fr) * 2019-12-13 2021-06-17 Inscripta, Inc. Nouvelles enzymes
WO2022097070A1 (fr) * 2020-11-04 2022-05-12 Specific Biologics Inc. Édition de gènes avec une endonucléase modifiée
JP2023548391A (ja) * 2020-11-04 2023-11-16 スペシフィック バイオロジクス インコーポレイテッド 改良型エンドヌクレアーゼによる遺伝子編集
WO2025046062A1 (fr) * 2023-08-31 2025-03-06 Snipr Biome Aps Nouveau type de système crispr/cas

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