Tnpb-based genome editors

Abstract

Provided herein are novel TnpB polypeptides having RNA-guided endonuclease activity. Also provided herein is a gene editing system comprising a TnpB polypeptide of the invention or an unarmed variant thereof, or a fusion polypeptide of identical content, and a method of gene editing with a TnpB polypeptide of the invention or an unarmed variant thereof, or a fusion polypeptide or gene editing system.

Description

Technical Field

[0001] This invention relates to molecular biology. In particular, this invention provides a novel RNA-guided system for gene editing. Background Technology

[0002] Using a site-specific system, the genome can be modified at predetermined sites.

[0003] Genome editing technologies such as large-scale nucleases, designed zinc finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs) can be used to generate targeted genome modifications, but these systems often have low specificity and require redesigned nucleases for each target site, making their preparation costly and time-consuming.

[0004] Recently, the CRISPR (clustered regularly spaced short palindromic repeats)-Cas system has become popular, containing different domains of effector proteins covering a variety of activities (DNA recognition, binding, and optional cleavage). However, Cas nucleases are generally large in size, making it difficult to deliver the CRISPR-Cas system into cells.

[0005] Transposition plays a crucial role in reshaping the genomes of all organisms.

[0006] The IS200 / IS605 and IS607 family of insertion sequences are among the simplest mobile genetic elements, containing only the genes necessary for their transposition and regulation. These elements encode the tnpA transposase, essential for movement, and typically carry a helper tnpB gene, which is dispensable for transposition. The TnpB protein (ISDra2 TnpB) has been reported to possess RNA-guided DNA endonuclease activity (Karvelis et al., Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease. Nature, 2021, 559:692-696). TnpB is the functional ancestor of CRISPR–Cas nucleases and has been established as a prototype for novel genome editing systems. TnpB proteins are generally much smaller than Cas proteins, thus facilitating delivery into cells.

[0007] However, ISDra2 TnpB requires recognition of the TTGAT transposon-associated motif (TAM) to achieve RNA-guided cleavage. Longer sequences are known to exist at a relatively low frequency in the genome. Therefore, the use of ISDra2 TnpB in genome editing is limited.

[0008] Therefore, there is a need to identify novel TnpB peptides with RNA-guided DNA endonuclease activity that recognize different TAMs, such as shorter TAMs, compared to ISDra2TnpB, and genome editing systems that contain these peptides. Summary of the Invention

[0009] To meet the aforementioned needs, the inventors identified numerous TnpB peptides with RNA-directed DNA endonuclease activity. The identification of TnpB peptides increases the possibility of editing various genomic regions that are inaccessible to existing TnpB peptides. Furthermore, the TnpB peptides disclosed herein offer higher editing efficiency than existing TnpB peptides, even comparable to Cas9 nucleases.

[0010] In a first aspect, this disclosure provides a recombinant gene editing system comprising

[0011] -TnpB polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding said TnpB polypeptide or a functional fragment thereof, and

[0012] Guide RNA (gRNA) contains a target region capable of hybridizing to a nucleotide sequence of interest and a backbone region capable of binding to a TnpB peptide or a functional fragment thereof, or a polynucleotide containing a nucleotide sequence encoding gRNA.

[0013] The TnpB polypeptide is derived from microorganisms selected from Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas media, Thermobifida fusca, Campylobacter coli, Synechococcus sp. JA-3-3Ab, Thermosynechococcuse longatus, Nostoc sp. PCC 7120, Clostridium botulinum type C, C-Stockholm phage c-st, and Methanosarcina mazei. The TnpB polypeptide contains *Acetivoxil*, *Escherichia coli*, *Salmonella enterica*, *Halorubrum halophilum*, *Klebsiella pneumoniae*, *Deinococcus geothermalis*, *Acinetobacter baumannii*, *Raoultella ornithinolytica*, *Anoxybacillus amylolyticus*, *Youngiibacter multivorans*, *Cytobacillus sp. CY-G*, *Clostridium vitabionis*, *Aeribacillus pallidus*, and *Bacillus thuringiensis*, and wherein the TnpB polypeptide has RNA-directed endonuclease activity.

[0014] In a second aspect, this disclosure provides a composition comprising:

[0015] - Recombinant TnpB peptide or its functional fragment

[0016] - Target double-stranded DNA containing the nucleotide sequence of interest and the TAM recognized by the TnpB peptide; and

[0017] - Recombinant guide RNA (gRNA) contains a targeting region capable of hybridizing to a nucleotide sequence of interest and a backbone region capable of binding to the TnpB peptide or a functional fragment thereof.

[0018] The TnpB polypeptide is derived from microorganisms selected from Enterococcus faecalis, Aeromonas salmonidae, Clostridium perfringens, Methanococcus masculinii, Bacillus cereus, Aeromonas intermedius, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc coli PCC 7120, Clostridium botulinum type C-Stockholm phage c-st, Methanococcus acetophilus, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Ornithine-lysinus, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, and Clostridium. The species include *Vitabionis*, *Bacillus pallida*, and *Bacillus thuringiensis*, and the TnpB polypeptide described therein has RNA-directed endonuclease activity.

[0019] In a third aspect, this disclosure provides a method for introducing a double-strand break into a polynucleotide of interest, comprising the step of contacting the polynucleotide with a recombinant gene editing system, the system comprising

[0020] -TnpB polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding said TnpB polypeptide or a functional fragment thereof, and

[0021] - Guide RNA (gRNA) contains a target region capable of hybridizing to a nucleotide sequence in a polynucleotide of interest and a backbone region capable of binding to a TnpB polypeptide or a functional fragment thereof, or a polynucleotide containing a nucleotide sequence encoding a gRNA.

[0022] The TnpB polypeptide is derived from microorganisms selected from *Enterococcus faecalis*, *Aeromonas salmonicida*, *Clostridium perfringens*, *Methanococcus martensii*, *Bacillus cereus*, *Aeromonas intermedius*, *Thermobifida fusca*, *Campylobacter coli*, *Synechococcus JA-3-3Ab*, *Thermosynechococcus elongatus*, *Nostoc* PCC 7120, *Clostridium botulinum* C-Stockholm phage c-st, *Methanococcus acetophilus*, *Escherichia coli*, *Salmonella enterica*, *Halorubrum halophilum*, *Klebsiella pneumoniae*, *Deinococcus geothermalis*, *Acinetobacter baumannii*, *Ornithine-lysinian* Raoultella, *Anoxybacillus amylolyticus*, *Youngiibacter multivorans*, *Cytobacillus sp. CY-G*, and *Clostridium*. The species include *Vitabionis*, *Bacillus pallida*, and *Bacillus thuringiensis*, and the TnpB polypeptide described therein has RNA-directed endonuclease activity.

[0023] In a fourth aspect, this disclosure provides a method for modifying a genomic sequence in a cell, comprising the step of introducing a recombinant gene editing system into the cell, the system comprising

[0024] -TnpB polypeptide or its functional fragment, or a polynucleotide containing a nucleotide sequence encoding a TnpB polypeptide or its functional fragment, and

[0025] Guide RNA (gRNA) consists of a target region capable of hybridizing to a portion of the genome sequence and a backbone region capable of binding to the TnpB peptide or a functional fragment thereof, or a polynucleotide containing a nucleotide sequence encoding the gRNA.

[0026] The TnpB polypeptide is derived from microorganisms selected from Enterococcus faecalis, Aeromonas salmonidae, Clostridium perfringens, Methanococcus masculinii, Bacillus cereus, Aeromonas intermedius, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc coli PCC 7120, Clostridium botulinum type C-Stockholm phage c-st, Methanococcus acetophilus, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Ornithine-lysinus, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, and Clostridium. The species include *Vitabionis*, *Bacillus pallida*, and *Bacillus thuringiensis*, and the TnpB polypeptide described therein has RNA-directed endonuclease activity.

[0027] In a fifth aspect, this disclosure provides a modified TnpB polypeptide that, compared to a parental TnpB polypeptide, contains a modification in the DDE motif, wherein the parental polypeptide has RNA-directed endonuclease activity, and wherein the modified TnpB loses its activity of cleaving double-stranded DNA.

[0028] In a sixth aspect, this disclosure provides a recombination system comprising:

[0029] - The fifth aspect refers to the modified TnpB polypeptide or its functional fragment, or a polynucleotide containing a nucleotide sequence encoding the modified TnpB polypeptide or its functional fragment, and

[0030] - Guide RNA (gRNA) contains a target region capable of hybridizing to a nucleotide sequence of interest and a backbone region capable of binding to a TnpB polypeptide or a functional fragment thereof, or a polynucleotide containing a nucleotide sequence encoding gRNA.

[0031] In a seventh aspect, this disclosure provides a method for modifying a genome sequence in a cell, comprising the step of introducing a recombination system of the sixth aspect and a gene editing system targeting the genome sequence into the cell, wherein the nucleotide sequence of interest is close to the genome sequence.

[0032] In an eighth aspect, this disclosure provides a fusion polypeptide comprising a TnpB polypeptide fused to a fusion partner or a functional fragment thereof, or a disarmed TnpB polypeptide, wherein the TnpB polypeptide is derived from a microorganism selected from Enterococcus faecalis, Aeromonas salmonidae, Clostridium perfringens, Methanococcus masculinii, Bacillus cereus, Aeromonas intermedius, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcuse longatus, Nostoc coli PCC 7120, Clostridium botulinum type C-Stockholm phage c-st, Methanococcus acetophilus, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Ornithine-lysinus, Anoxybacillus amylolyticus, Youngiibacter multivorans, and Cytobacillus. sp. CY-G, Clostridium vitabionis, Bacillus pallida and Bacillus thuringiensis, wherein the TnpB polypeptide has RNA-directed endonuclease activity, and wherein the disarmed TnpB polypeptide loses its activity of cleaving double-stranded RNA.

[0033] In a ninth aspect, this disclosure provides a gene editing system comprising

[0034] -The fusion polypeptide disclosed herein, or a polynucleotide comprising a nucleotide sequence encoding said fusion polypeptide, and

[0035] - Guide RNA (gRNA) contains a target region capable of hybridizing to a nucleotide sequence of interest and a backbone region capable of binding to a TnpB polypeptide or a functional fragment thereof, or a polynucleotide containing a nucleotide sequence encoding gRNA.

[0036] In a tenth aspect, this disclosure provides a method for modifying a genome sequence in a eukaryotic cell, comprising the step of introducing a gene editing system of the ninth aspect into the eukaryotic cell, wherein the gRNA contains a target region capable of hybridizing with a portion of the genome sequence.

[0037] In the eleventh aspect, this disclosure provides a method for screening the activity of TnpB peptides in cleaving double-stranded DNA, comprising the following steps:

[0038] - Provides candidate TnpB peptides from microorganisms;

[0039] - Provides a gRNA containing a target region and a backbone region, wherein the backbone region contains 100-350 nucleotides prior to the 3' end of the IS, which naturally contains a nucleotide sequence encoding the TnpB polypeptide;

[0040] - Provide target DNA containing a nucleotide sequence that hybridizes to the nucleotide sequence of the target region and a TAM recognized by the TnpB peptide, wherein the TAM consists of four or five consecutive nucleotides adjacent to the 5' end of IS.

[0041] - Enables the TnpB peptide to contact gRNA and target DNA; and

[0042] - Detect cuts on the target DNA. Attached Figure Description

[0043] Figure 1 This image shows a map of a pair of plasmids used for screening TnpB peptides with RNA-directed DNA endonuclease activity, including the test plasmid (encoding the TnpB peptide and gRNA). Figure 1 A) and reporter plasmids (containing target sequences, Figure 1 B).

[0044] Figure 2 The results of screening for TnpB peptides with RNA-directed DNA endonuclease activity are shown.

[0045] Figure 3 The loss ratio of TnpB peptide and dTnpB peptide (TnpB peptide with DDE motif replaced by alanine) is shown.

[0046] Figure 4 The amino acid sequence alignment of the TnpB polypeptide with RNA-directed DNA endonuclease activity is shown.

[0047] Figure 5 The amino acid sequence of the TnpB polypeptide with RNA-directed DNA endonuclease activity is shown, along with the DDE motif (residues in bold and underline) within the amino acid sequence.

[0048] Figure 6 The structure and mechanism of the fluorescence reporter system are shown.

[0049] Figure 7 The image shows a plasmid map of RNA-guided cleavage detected in 293T cells using a fluorescent reporter system, including a plasmid (A) encoding the fluorescent reporter system, a plasmid (B) encoding the TnpB polypeptide, and a plasmid (C) encoding gRNA.

[0050] Figure 8The flow cytometry results used to detect GFP expression are shown, demonstrating RNA-guided cleavage in the fluorescent reporter system caused by peptides ISTfu1TnpB, ISDge10 TnpB, ISAba30 TnpB, ISAam1, and ISYmu1 TnpB.

[0051] Figure 9 The editing efficiency of ISTfu1 TnpB and ISDra2 TnpB peptides with different TAMs is shown in the fluorescence reporter system.

[0052] Figure 10 The results of a surveyor assay for RNA-directed cleavage of ISTfu1 TnpB (Fig. A), ISDge10 TnpB (Fig. B), ISAba30 TnpB (Fig. C), ISAam1 (Fig. D), and ISYmu1 (Fig. E) TnpB peptides in human cells are shown.

[0053] Figure 11 The effects of backbone design on RNA-guided cleavage of ISDra2 (Fig. A), ISTfu1 (Fig. B), ISDge10 (Fig. C), ISAba30 (Fig. D), ISAam1 (Fig. E), and ISYmu1 (Fig. F) TnpB peptides are shown.

[0054] Figure 12 The distribution of 10 conserved residues in 25 active TnpB proteins and ISDra2 TnpB is shown in Figure A, which are marked with asterisks and black lines in the bottom column, superimposed domain structures, and the endonuclease activity of TnpB mutants (N to A) is sharply reduced (Figure B).

[0055] Figure 13 The editing efficiency of ISAam1, ISYmu1, and ISDra2 at six randomly selected endogenous sites in HEK293T cells is shown. Data are presented as mean ± SD, n = 3.

[0056] Figure 14 This diagram shows the gRNA designs for seven nucleases at 10 genomic loci in the human genome. The nucleases and their corresponding TAMs are color-coded. The gRNAs are aligned according to strand position. For example, in the CBLB example, the gRNAs for ISAam1 and three Cas12f variants overlap more than the other three nucleases.

[0057] Figure 15The figures show a comparison of editing efficiencies of two TnpB systems and five Cas nucleases at 10 genomic loci in human HEK293T (Figure A) and HCT116 (Figure B) cells, and a comparison of editing efficiencies of ISAam1 (Figure C) or ISYmu1 (Figure D) relative to the five Cas nucleases at 3 genomic loci in HEK293T cells. For Figures A and B, each point represents the average efficiency of three biological replicates. The distribution is shown as a box plot, where the boxes represent the median (middle line) and interquartile range (IQR, box boundaries), indicated by whisker lines from minimum to maximum. For Figures C and D, gRNA designs are shown on the left and editing efficiencies on the right. The seven nucleases are color-coded. Since it is impossible to design overlapping gRNAs targeting the same sites in all seven nucleases, two sets of overlapping gRNAs were designed for ISAam1, the three Cas12f variants and Nme2-C.NR, and ISAam1 and SaCas9, respectively. ISYmu1 is in a similar situation.

[0058] Figure 16 The editing efficiency of Rosa26 site based on AAV2 delivery in mouse C2C12 cells is shown (n=3, Fig. A), and the in vivo gene editing efficiency of Rosa26 site (n=4) and Angptl3 site (n=3) mediated by AAV8 in mice is shown (Fig. B). Data are presented as mean ± SD.

[0059] Figure 17 The off-target to on-target editing efficiency ratios for the first two predicted off-target sites of each nuclease are shown (n=3, Figure A), as well as the off-target level quantified by iGUIDE analysis at the MAPK8 site in HEK293T cells (Figure B, where pie charts show the ratios of on-target and off-target readings, respectively). Invention Details

[0061] 1 Definition

[0062] As used herein, the term "TnpB polypeptide" refers to a polypeptide encoded by the tnpB gene in the inserted sequence (IS). "TnpB endonuclease," "TnpB effector," and "TnpB nuclease" are used interchangeably herein to refer to TnpB polypeptides with RNA-directed endonuclease activity. TnpB polypeptides are generally 300-500 amino acid residues in size. TnpB polypeptides "derived from" microorganisms refer to naturally occurring TnpB polypeptides in microorganisms, including TnpB polypeptides and their natural variants that can be found in online databases such as the National Center for Biotechnology Information (NCBI).

[0063] The terms “peptide” and “protein” are used interchangeably in this document and refer to polymers of amino acids, including full-length proteins and their fragments.

[0064] Endonucleases are enzymes that cleave phosphodiester bonds within polynucleotide chains, including restriction endonucleases that cleave DNA at specific sites without damaging bases. Examples of endonucleases include, but are not limited to, restriction endonucleases, broad-spectrum nucleases, TAL-effect nucleases (TALENs), zinc finger nucleases, and Cas (CRISPR-associated) effector endonucleases. This disclosure provides a novel RNA-directed TnpB endonuclease.

[0065] As used herein, “nucleic acid” refers to a polynucleotide, a single- or double-stranded polymer comprising deoxyribonucleotide or ribonucleotide bases. Nucleic acids may also include fragments and modified nucleotides. Therefore, the terms “polynucleotide,” “nucleic acid sequence,” “nucleotide sequence,” and “nucleic acid fragment” are used interchangeably to refer to single- or double-stranded RNA and / or DNA and / or RNA-DNA polymers, optionally containing synthetic, non-natural, or modified nucleotide bases. Nucleotides (usually present as 5'-monophosphates) are designated by their single-letter names as follows: “A” for adenosine or deoxyadenosine (for RNA or DNA, respectively), “C” for cytosine or deoxycytosine, “G” for guanosine or deoxyguanosine, “U” for uridine, “T” for deoxythymidine, “R” for purine (A or G), “Y” for pyrimidine (C or T), “K” for G or T, “H” for A, C, or T, “I” for inosine, and “N” for any nucleotide.

[0066] The term "genome," applicable to prokaryotic and eukaryotic cells or somatic cells of organisms, includes not only chromosomal DNA within the cell nucleus but also organelle DNA within subcellular components of the cell (e.g., mitochondria or plasmids). The term "genome" refers to the complete complement of the genetic material (genes and non-coding sequences) present in every cell, virus, or organelle of an organism; and / or a complete set of chromosomes inherited as (haploid) units from one parent.

[0067] The term "selective hybridization" refers to the hybridization of a nucleic acid sequence with a specific target nucleic acid sequence, preferably under stringent hybridization conditions, to a level that is more detectable than hybridization with non-target nucleic acid sequences (e.g., at least twice the background level), and substantially excludes non-target nucleic acids. Selective hybridization sequences typically have at least 80% sequence identity, or 90% sequence identity, and up to 100% sequence identity (i.e., complete complementarity) with each other.

[0068] The term "strict conditions" or "strict hybridization conditions" refers to the conditions under which a probe selectively hybridizes with its target sequence in an in vitro hybridization assay. Strict conditions are sequence-dependent and vary under different conditions. By controlling the strictness of hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous detection). Alternatively, strict conditions can be adjusted to allow some mismatches in the sequences, thereby detecting a lower degree of similarity (heterologous detection). Various hybridization conditions, including strict hybridization conditions and highly strict hybridization conditions, are known to those skilled in the art. See, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, John Wiley & Sons, NY.

[0069] The term "homology" refers to similar DNA sequences. For example, a "region homologous to a genomic region" found on donor DNA is a DNA region that has a similar sequence to a given "genomic region" in the genome of a cell or organism. Homologous regions can be of any length, as long as they are long enough to facilitate homologous recombination at the cleavage target site. For example, homologous regions can contain 5 to 3000 or more bases, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000 bases, to achieve homologous recombination with the corresponding genomic region.

[0070] As used in this article, a “genomic region” is a segment of DNA on a cell’s chromosome or organelle that is located upstream or downstream of a target site, or that contains a portion of the target site (at the 5’ or 3’ end). Genomic regions can contain 5 to 3000 or more bases, for example, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000 bases in length, to enable homologous recombination with corresponding homologous regions.

[0071] The term "homologous recombination" (HR) refers to the exchange of DNA fragments between two DNA molecules at homologous sites. The frequency of homologous recombination is influenced by many factors. The amount of homologous recombination and the relative ratio of homologous to non-homologous recombination vary among different organisms. Generally, the length of the homologous region affects the frequency of homologous recombination events: the longer the homologous region, the higher the frequency. Furthermore, homologous recombination requires a certain length of homologous region, which is species-variable. See, for example, Singer et al., (1982) Cell 31:25-33; Shen and Huang, (1986) Genetics 112:441-57; Watt et al., (1985) Proc.Natl.Acad.Sci.USA82:4768-72, Sugawara and Haber, (1992) Mol CellBiol12:563-75, Rubnitz and Subramani, (1984) Mol Cell Biol 4:2253-8; Ayares et al., (1986) Proc. Natl. Acad. Sci. USA 83:5199-203; Liskay et al., (1987) Genetics 115:161-7.

[0072] "Sequence identity" or "identity" in nucleotide or amino acid sequences means that the nucleotide bases or amino acid residues in two sequences are identical when compared with maximum correspondence in a specified comparison window.

[0073] The term "sequence identity percentage" refers to a value determined by comparing two best-aligned sequences within a comparison window. This comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which contains no additions or deletions) to achieve optimal alignment. The sequence identity percentage is calculated by dividing the number of matching positions (i.e., positions where nucleotide bases or amino acid residues in both sequences are the same) by the total number of positions in the comparison window and multiplying the result by 100. For example, when aligning two sequences, if 950 of the best-aligned positions in a comparison window of 1000 positions are the same, then the sequences are 95% identical to each other.

[0074] Various comparison methods have been designed for sequence alignment and calculation of the percentage of identical or similar sequences, including but not limited to the MegAlign™ program of the LASERGENE Bioinformatics Computing Suite (DNASTAR Inc., Madison, Wis.). In the context of this application, it should be understood that when performing analysis using sequence analysis software, unless otherwise stated, the analysis results will be based on the “default values” of the cited program. As used herein, “default values” refers to any set of values ​​or parameters initially loaded with the software during initialization.

[0075] BLAST is a search algorithm provided by NCBI for finding similar regions between biological sequences. The program compares nucleotide or protein sequences to a sequence database and calculates the statistical significance of matches to identify sequences that are sufficiently similar to the query sequence to avoid predicting that the similarity is random. BLAST reports the identified sequences and their local alignments with the query sequence. Those skilled in the art will well understand that many levels of sequence identity can be used to identify polypeptides from other species or naturally or synthetically modified polypeptides that have the same or similar functions or activities. Useful examples of identity percentages include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any percentage from 50% to 100%. In fact, any amino acid identity from 50% to 100% can be used to describe this disclosure, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0076] A "centimolecular unit" (cM) or "mapping unit" is the distance between two polynucleotide sequences, linked genes, markers, target sites, genomes, or any pair thereof, where 1% of the meiotic products are recombinations. Therefore, one cM is equivalent to the distance between two linked genes, markers, target sites, loci, or any pair thereof that equals the average recombination frequency of 1%.

[0077] "Isolated" polynucleotides, peptides, or proteins are substantially or essentially free of components that are typically associated with or interact with polynucleotides, peptides, or proteins and are found in their natural environment. Therefore, isolated polynucleotides, peptides, or proteins are substantially free of other cellular material or culture media when prepared by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, "isolated" polynucleotides are free of sequences naturally flanking the polynucleotides in the genomic DNA of the organism from which they are derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide). Isolated polynucleotides and peptides can be purified from the cells in which they are naturally present. Methods for isolating or purifying polynucleotides or peptides are known to those skilled in the art. The term also covers recombinant or chemically synthesized polynucleotides and peptides.

[0078] The term "fragment" refers to a sequence of consecutive nucleotides or amino acids. In one embodiment, the fragment comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides. In another embodiment, the fragment comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive amino acids. The fragment may or may not exhibit the function of a sequence having a certain percentage of identity over the fragment length.

[0079] The term "functional fragment" refers to a portion of an isolated polynucleotide or polypeptide that exhibits the same activity or function as its source, either the longer or full-length sequence.

[0080] The term "gene" includes nucleic acid fragments that express functional molecules, such as, but not limited to, specific proteins, including regulatory sequences before (5' non-coding sequence) and after (3' non-coding sequence).

[0081] The term "endogenous" refers to sequences or other molecules that are naturally present in cells or organisms. Endogenous polynucleotides are typically found within the cell's genome; that is, they are not heterologous.

[0082] The term "heterologous" refers to the difference between the original environment, location, or composition of a particular polynucleotide or polypeptide and its current environment, location, or composition. Non-limiting examples include taxonomic differences (e.g., a polynucleotide obtained from species A is heterologous if inserted into the genome of species B, or into the genome of a different variety or cultivar of species A; or a polynucleotide obtained from bacteria introduced into the cells of a plant or animal), or sequence differences (e.g., a polynucleotide obtained from species A, isolated, modified from a plant reintroduced into species A).

[0083] An "allele" is one of several alternative forms of a gene that occupies a given locus on a chromosome. A cell or organism is homozygous at a given locus when all alleles at that locus are identical. A cell or organism is heterozygous at a given locus if the alleles at that locus are different.

[0084] A "coding sequence" refers to a nucleotide sequence that encodes a specific amino acid sequence. A "regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequence), inside, or downstream (3' non-coding sequence) of a coding sequence, which affects the transcription, RNA processing, stability, or translation of the related coding sequence. Regulatory sequences include, but are not limited to, promoters, pretranslational sequences, 5' non-translated sequences, 3' non-translated sequences, introns, polyadenylation signal sequences, RNA processing sites, effector binding sites, and stem-loop structures.

[0085] A "mutated gene" is a gene altered through human intervention. The sequence of a "mutated gene" differs from that of a corresponding non-mutated gene, with at least one nucleotide added, deleted, inserted, or substituted. In this disclosure, a mutant gene comprises alterations caused by the guide polynucleotide / TnpB endonuclease system disclosed herein. A mutant organism is an organism containing a mutant gene.

[0086] As used herein, “targeted mutation” is a gene mutation produced in a target sequence within a gene using any method known to those skilled in the art, including the methods disclosed herein involving the guidance of the TnpB endonuclease system.

[0087] The term "knockout" refers to the partial or complete invalidation of a DNA sequence in a cell, for example, by targeting the TnpB protein of this disclosure; for example, the DNA sequence before knockout may encode an amino acid sequence or may have a regulatory function (e.g., a promoter).

[0088] The term "knock-in" refers to the replacement or insertion of a DNA sequence at a specific site in the cell's genome by targeting with the TnpB protein (e.g., via homologous recombination (HR), in which a suitable donor DNA polynucleotide is also used). Knock-in can be a specific insertion of a heterologous nucleotide sequence encoding an amino acid sequence or functional RNA, or a specific insertion of a transcriptional regulatory element.

[0089] The term "domain" refers to a continuous nucleotide (which can be RNA, DNA, and / or RNA-DNA combination sequences) or a continuous or discontinuous amino acid sequence.

[0090] A "conserved domain" or "motif" refers to a group of nucleotides or amino acids that are conserved at a specific position in the aligned sequence of an evolutionarily related gene or protein. While nucleotides or amino acids at other positions can vary between homologous proteins, highly conserved nucleotides or amino acids at specific positions represent amino acids that are essential for the structure, stability, or function of a polynucleotide or protein.

[0091] "Codon-optimized" nucleotide sequences are nucleotide sequences whose codon usage frequencies are designed to mimic the preferred codon usage frequencies of the host cell. "Optimized" polynucleotides include nucleotide sequences optimized for improved expression in specific heterologous host cells.

[0092] A promoter is a nucleotide sequence that participates in the recognition and binding of RNA polymerases and other proteins to initiate transcription. Promoters can be derived entirely from natural genes, or consist of different elements derived from different promoters found in nature, and / or contain synthetic DNA fragments. Those skilled in the art will understand that different promoters can direct gene expression in different tissues or cell types, at different developmental stages, or under different environmental conditions. It is further recognized that, since the exact boundaries of the regulatory sequence are not fully determined in most cases, certain variant DNA fragments may possess the same promoter activity.

[0093] Promoters that result in gene expression in most tissues or cell types most of the time are generally called "constitutive promoters." The terms "inducible promoters" or "regulatory promoters" refer to promoters that selectively express coding sequences or functional RNA in response to the presence of endogenous or exogenous stimuli, such as compounds (chemical inducers), or in response to environmental, hormonal, chemical, and / or developmental signals. Inducible or regulatory promoters include, for example, promoters induced or regulated by light, heat, stress, flood or drought, salt stress, osmotic stress, plant hormones, damage, or chemicals such as ethanol, abscisic acid (ABA), jasmonic acid, salicylic acid, or safeners.

[0094] An enhancer is a nucleotide sequence that can stimulate promoter activity. It can be an intrinsic element of the promoter or a heterologous element inserted to enhance promoter activity or tissue specificity.

[0095] The term "leader sequence" refers to the nucleotide sequence located between the promoter and coding sequences. Leader sequences are present in mRNA upstream of the start codon. Leader sequences can influence the processing of the initial transcript into mRNA, mRNA stability, or translation efficiency.

[0096] The term "3' non-coding sequence" can be interchanged with "transcription terminator" or "termination sequence" and refers to a nucleotide sequence located downstream of a coding sequence, including polyadenylation signaling sequences and other sequences encoding regulatory signals that can affect mRNA processing or gene expression. Polyadenylation signals are typically characterized by influencing the addition of polyadenylate fragments to the 3' end of mRNA precursors.

[0097] The term "RNA transcript" refers to the product of transcription of a DNA sequence catalyzed by RNA polymerase. When the RNA transcript is a perfectly complementary copy of the DNA sequence, it is called a primary transcript or pre-mRNA. RNA transcripts derived from pre-mRNA after post-transcriptional processing are called mature RNA or messenger RNA (mRNA). "Messenger RNA" or "mRNA" refers to RNA that can be translated into protein and does not contain introns. "cDNA" refers to DNA that is complementary to an mRNA template and synthesized from it using reverse transcriptase. cDNA can be single-stranded or converted into a double-stranded form using, for example, the Klenow fragment of DNA polymerase I. "Sense" RNA refers to RNA transcripts that include mRNA and can be translated into protein, either intracellularly or in vitro. "Antisense RNA" refers to RNA transcripts that are fully or partially complementary to a target primary transcript or mRNA and can block the expression of a target gene. The complementarity of antisense RNA can be with any part of a specific gene transcript, i.e., in the 5' untranslated region, 3' untranslated region, intron, or coding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but affects cellular processes. The terms “complementary” and “anticomplementary” are used interchangeably in relation to mRNA transcripts and are intended to define the information-providing antisense RNA.

[0098] The term "operably linked" refers to the association of nucleotide sequences on a single nucleic acid fragment, thereby allowing the function of one to be regulated by the other. For example, when a promoter can regulate the expression of a coding sequence, the promoter and coding sequence are operably linked (i.e., the coding sequence is transcribed under the control of the promoter). The coding sequence can be operably linked to the regulatory sequence in either the sense or the antisense direction.

[0099] The term "host" refers to an organism or cell in which a heterologous component (polynucleotide, polypeptide, other molecule, cell) has been introduced. As used herein, "host cell" refers to a cell in vivo or isolated from a eukaryotic cell, prokaryotic cell (e.g., bacterial or archaea cell), or a cell of a multicellular organism cultured as a single-celled entity (e.g., a cell line) in which a heterologous polynucleotide or polypeptide has been introduced. The cells are selected from: archaea cells, bacterial cells, eukaryotic cells, eukaryotic single-celled organisms, somatic cells, germ cells, stem cells, plant cells, algal cells, and animal cells, such as invertebrate cells, vertebrate cells, fish cells, frog cells, avian cells, insect cells, mammalian cells, pig cells, bovine cells, goat cells, sheep cells, rodent cells, rat cells, mouse cells, non-human primate cells, and human cells. In some cases, the cells are isolated. In some cases, the cells are in vivo.

[0100] The term "recombination" refers to the artificial combination of two originally separate sequence fragments, for example, through chemical synthesis or through genetic engineering techniques.

[0101] The terms "plasmid" and "vector" refer to linear or circular extrachromosomal elements that typically carry genes not metabolized at the center of the cell and are usually present as double-stranded DNA. These elements can be autonomously replicating sequences, genome-integrated sequences, bacteriophages, or nucleotide sequences derived from any source of single-stranded or double-stranded DNA or RNA, existing in linear or circular forms, where many nucleotide sequences have been linked or recombined into unique structures capable of introducing polynucleotides of interest into the cell.

[0102] When referring to nucleic acid molecules, the term "construct" includes an artificial combination of nucleic acid sequences, such as regulatory and coding sequences that are not all present simultaneously in nature. When a nucleic acid construct contains control sequences required to express the coding sequences of the present invention, the term is synonymous with the term "expression cassette." For example, a nucleic acid construct may contain regulatory and coding sequences derived from different sources, or regulatory and coding sequences derived from the same source but arranged in a manner different from those found in nature. Such constructs can be used alone or in combination with a vector. If a vector is used, the choice of vector depends on the method of introducing the vector into a host cell, as is well known to those skilled in the art. A vector used to express a coding sequence (e.g., containing an expression construct) is called an "expression vector."

[0103] As used herein, the term “expression” refers to the production of a functional end product (e.g., mRNA, guide RNA, or protein) in its precursor or mature form.

[0104] "Mature" proteins refer to post-translational processed polypeptides (i.e., polypeptides in which any precursor peptides or precursor peptides present in the primary translation product have been removed).

[0105] "Precursor" proteins refer to the primary products of mRNA translation (i.e., the propeptide and precursor peptide are still present). Propeptides and precursor peptides can be, but are not limited to, intracellular localization signals.

[0106] As used herein, an "effectant" or "effectant protein" is a protein having activities including recognizing, binding to, and / or cleaving or splitting polynucleotide targets. An effectant or effectant protein can also be an endonuclease, such as the TnpB polypeptide of this invention. The "effect complex" of the gene editing system includes the TnpB polypeptide involved in gRNA and target recognition and binding.

[0107] The “functional fragment” of a TnpB endonuclease refers to a portion of the TnpB endonuclease disclosed herein that retains the ability to recognize, bind to, and / or cleave (introducing double-strand breaks) target sites. The “functional variant” of a TnpB endonuclease refers to a variant of the TnpB endonuclease disclosed herein that retains the ability to recognize, bind to, and / or cleave target sequences.

[0108] TnpB endonucleases may also include multifunctional TnpB endonucleases, which are single polypeptides having endonuclease activity (containing at least one protein domain that can act as an endonuclease) and at least one other function, such as, but not limited to, the function of forming a complex (containing at least one second protein domain that can form a complex with other proteins).

[0109] As used herein, the term "guide polynucleotide" refers to a polynucleotide that can form a complex with a TnpB endonuclease, as described herein, and enable the Cas endonuclease to recognize, optionally bind to, and optionally cleave DNA target sites. The guide polynucleotide can be a guide RNA, a guide DNA sequence, or a combination thereof (RNA-DNA combinatorial molecule). For TnpB polypeptides, the guide RNA is also referred to as "right-element RNA" or "reRNA".

[0110] A “functional fragment” of a guide polynucleotide refers to a portion or subsequence of the guide polynucleotide of this disclosure, wherein the ability to function as a guide polynucleotide is retained. A “functional variant” of a guide polynucleotide refers to a variant of the guide polynucleotide of this disclosure, wherein the ability to function as a guide polynucleotide is retained.

[0111] The terms “targeting domain” and “targeting region” are used interchangeably in this document and include nucleotide sequences that can hybridize (complement) one strand (nucleotide sequence) of a double-stranded DNA target site. The percentage of complementarity between the target region and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The length of the variable targeting region can be at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the variable targeting domain comprises a continuous segment of 12-30 nucleotides. The targeting domain can consist of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence, or any combination thereof.

[0112] The "backbone" of the guide polynucleotide contains a nucleotide sequence that interacts with the TnpB polypeptide.

[0113] The term "gRNA / TnpB complex" refers to the RNA component and TnpB endonuclease that can form a complex, wherein the complex can guide the TnpB endonuclease to the DNA target site, enabling the TnpB endonuclease to recognize, bind to and / or cleave (introduce double-strand breaks) the DNA target site.

[0114] The terms “target site,” “target sequence,” and “target region” are used interchangeably herein and refer to a nucleotide sequence (including chromosomal, chloroplast, mitochondrial DNA, and plasmid DNA) on a chromosome, free organism, locus, or any other DNA molecule in the cellular genome that the gRNA / TnpB complex can recognize, bind to, and optionally cleave or cut. A target site can be an endogenous site in the cellular genome, or it can be heterologous to the cell and thus not naturally present in the cellular genome, or it can be found at a heterologous genomic location compared to its naturally occurring location.

[0115] In this article, "transposon-associated motif" refers to a short nucleotide sequence adjacent to the target sequence recognized (targeted) by the gRNA / TnpB complex described herein. If the target DNA sequence is not adjacent to the TAM sequence, the TnpB endonuclease may fail to recognize the target DNA sequence. The sequence and length of the TAM in this article can vary depending on the TnpB protein. The TAM sequence is typically 4 or 5 nucleotides long.

[0116] "Modified" TnpB peptides / endonucleases refer to TnpB peptides that contain at least one amino acid substitution, deletion, insertion, or addition compared to naïve or wild-type TnpB peptides. If a modified TnpB peptide loses its DNA-cutting activity but retains its ability to recognize and bind polynucleotides, it can be called a "disarmed" TnpB peptide.

[0117] The terms “altered target site,” “altered target sequence,” “modified target site,” and “modified target sequence” are used interchangeably herein and refer to a target sequence as disclosed herein that contains at least one alteration compared to an unaltered target sequence. Such “alteration” includes, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, (iv) chemical alteration of at least one nucleotide, or (v) any combination of (i)-(iv).

[0118] "Modified nucleotide" or "edited nucleotide" refers to a nucleotide sequence of interest that contains at least one alteration compared to its unmodified nucleotide sequence. Such "alterations" include, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, (iv) chemical change of at least one nucleotide, or (v) any combination of (i)-(iv).

[0119] The terms "modifying target sites" and "changing target sites" are used interchangeably in this article, referring to methods that produce altered target sites.

[0120] As used herein, “donor DNA” is a DNA construct containing the polynucleotide of interest to be inserted into the gRNA / TnpB complex target site of the present invention.

[0121] The term "polynucleotide modification template" includes a polynucleotide containing at least one nucleotide modification compared to the nucleotide sequence to be edited. A nucleotide modification can be a substitution, addition, insertion, or deletion of at least one nucleotide. Optionally, the polynucleotide modification template may further include homologous nucleotide sequences flanking the at least one nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology to the desired nucleotide sequence to be edited.

[0122] As used herein, regarding sequence position, the term "before" means that one sequence appears upstream of another sequence (at the 5' end for nucleotide sequences and at the N-end for amino acid sequences). Regarding sequence position, the term "after" means that one sequence appears downstream of another sequence (at the 3' end for nucleotide sequences and at the C-end for amino acid sequences).

[0123] 2. TnpB polypeptide with RNA-directed endonuclease activity

[0124] The inventors identified several novel RNA-directed endonucleases encoded by the TnpB gene in the insert sequence (IS), specifically the TnpB polypeptide. This means the TnpB polypeptide can function as an effector protein in gene editing systems. Sequence analysis revealed that the active TnpB polypeptide contains an N-terminal helical-turn-helical (HTH) domain, a central domain, a C-terminal zinc finger domain, and a DDE motif.

[0125] Therefore, this disclosure provides an isolated TnpB polypeptide or a functional fragment thereof having RNA-directed endonuclease activity. In some embodiments, the TnpB polypeptide comprises an N-terminal HTH domain, a central domain, a C-terminal zinc finger domain, and a DDE motif.

[0126] In some embodiments, the TnpB polypeptide is derived from microorganisms selected from Enterococcus faecalis, Aeromonas salmonidae, Clostridium perfringens, Methanococcus martensii, Bacillus cereus, Aeromonas intermedius, Thermobifidafusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc coli PCC7120, Clostridium botulinum type C-Stockholm phage c-st, Methanococcus acetophilus, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Ornithine-lysinic Raoultella, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Airborne Bacillus pallida, and Bacillus thuringiensis.

[0127] In some embodiments, the TnpB polypeptide is encoded by the tnpB gene in an insert sequence (IS) selected from ISEfa4, ISAs26, ISCpe2, ISMma22, ISBce3, ISAeme8, ISTfu1, ISCco1, ISSoc3, ISTel2, ISNsp3, ISCbt1, ISMac7, ISEc46, ISSen6, ISAhl1, ISKpn69, ISDge10, ISKpn85, ISNsp2, ISAba30, ISRor9, ISAam1, ISYmu1, ISCytsp1, ISCvi1, ISCvi2, ISAepa1, and ISBth16.

[0128] In some embodiments, the TnpB polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the TnpB polypeptide is composed of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.

[0129] In some embodiments, the TnpB polypeptide is a functional variant comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the TnpB polypeptide is a functional variant consisting of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. The variants may differ from SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 in that one or more amino acids are substituted, inserted, deleted, and / or added. In some embodiments, the variants are conserved at the positions corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO:7.

[0130] In some embodiments, the functional fragment comprises at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the functional fragment consists of at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.

[0131] In some embodiments, the TnpB peptide or a functional fragment thereof recognizes a TAM adjacent to the nucleotide sequence of interest and has endonuclease activity. Compared to the ISDra2 TnpB peptide, the TnpB peptide of this disclosure can recognize shorter TAMs. In some embodiments, the TnpB peptide of this disclosure can recognize a TAM consisting of four consecutive nucleotides.

[0132] In some embodiments, the TnpB peptide of this disclosure can recognize the TAM of CCAT, CTAC, TGAC, TGAT, TTAC, TTAG, TTAA, TTAT, ACAT, TTTAT, TTTAA, or TTGAT.

[0133] In some embodiments, the TnpB peptide recognizes the TAM of CCAT. In some embodiments, the TnpB peptide comprises amino acids of SEQ ID NO: 2, 9, 15, 17, or 19.

[0134] In some embodiments, the TnpB peptide recognizes the TAM of CTAC. In some embodiments, the TnpB peptide comprises the amino acid of SEQ ID NO:16.

[0135] In some embodiments, the TnpB peptide recognizes the TAM of TGAY, where Y = C or T. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 7, 10, or 21. In some embodiments, the TnpB peptide recognizes the TAM of TGAC. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 10 or 21. In some embodiments, the TnpB peptide recognizes the TAM of TGAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 7.

[0136] In some embodiments, the TnpB peptide recognizes the TAM of TTAN, where N is any nucleotide. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, 8, 11, 12, 13, 14, 18, 20, 22, 26, 28, and 29. In some embodiments, the TnpB peptide recognizes the TAM of TTAC. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 5, 11, 12, or 20. In some embodiments, the TnpB peptide recognizes the TAM of TTAG. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 4, 6, or 14. In some embodiments, the TnpB peptide recognizes the TAM of TTAA. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 1, 22, 28, or 29. In some embodiments, the TnpB peptide recognizes the TAM of TTAT. In some embodiments, the TnpB polypeptide comprises an amino acid sequence of SEQ ID NO: 8, 13, 18 or 26.

[0137] In some embodiments, the TnpB peptide recognizes the TAM of TTTAW, where W = A or T. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TnpB peptide recognizes the TAM of TTTAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:23. In some embodiments, the TnpB peptide recognizes the TAM of TTTAA. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:25 or 27.

[0138] In some embodiments, the TnpB peptide recognizes the TAM of TTGAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:24.

[0139] In some embodiments, the TnpB polypeptide or a functional fragment thereof disclosed herein is capable of RNA-directed cleavage in prokaryotic and / or eukaryotic cells, preferably in prokaryotic and eukaryotic cells. In some embodiments, the TnpB polypeptide comprises the amino acid sequence of SEQ ID NO: 7, 18, 21, 23, or 24. In some embodiments, the TnpB polypeptide consists of the amino acid sequence of SEQ ID NO: 7, 18, 21, 23, or 24.

[0140] In some embodiments, the TnpB polypeptide is a functional variant comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:7, 18, 21, 23, or 24. In some embodiments, the TnpB polypeptide is a functional variant composed of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:7, 18, 21, 23, or 24. The variants may differ from SEQ ID NO:7, 18, 21, 23, or 24 in that they involve the substitution, insertion, deletion, and / or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some embodiments, the positions of the variants corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO:7 are conserved.

[0141] In some embodiments, the functional fragment comprises at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 7, 18, 21, 23, or 24. In some embodiments, the functional fragment consists of at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 7, 18, 21, 23, or 24.

[0142] Examples of DDE motifs include

[0143] D186, E280, and D362 of SEQ ID NO:1;

[0144] D188, E272 and D368 of SEQ ID NO:2;

[0145] D205, E289, and D372 of SEQ ID NO:3;

[0146] D184, E268, and D348 of SEQ ID NO:4;

[0147] D175, E260, and D339 of SEQ ID NO:5;

[0148] D186, E270, and D352 of SEQ ID NO:6;

[0149] D181, E265, and D361 of SEQ ID NO:7;

[0150] D199, E293, and D383 of SEQ ID NO:8;

[0151] D211, E295, and D373 of SEQ ID NO:9;

[0152] D185, E268, and D350 of SEQ ID NO:10;

[0153] D181, E265, and D345 of SEQ ID NO:11;

[0154] D234, E342, and D436 of SEQ ID NO:12;

[0155] D184, E268, and D350 of SEQ ID NO:13;

[0156] D185, E269, and D351 of SEQ ID NO:14;

[0157] D189, E273, and D369 of SEQ ID NO:15;

[0158] D190, E290, and D376 of SEQ ID NO:16;

[0159] D188, E272, and D368 of SEQ ID NO:17;

[0160] D187, E271, and D351 of SEQ ID NO:18;

[0161] D188, E272, and D368 of SEQ ID NO:19;

[0162] D181, E265, and D345 of SEQ ID NO:20;

[0163] D184, E268, and D364 of SEQ ID NO:21;

[0164] D183, E267, and D349 of SEQ ID NO:22;

[0165] D186, E270, and D351 of SEQ ID NO:23;

[0166] D185, E279, and D361 of SEQ ID NO:24;

[0167] D188, E272 and D352 of SEQ ID NO:25;

[0168] D181, E265, and D348 of SEQ ID NO:26;

[0169] D186, E276, and D359 of SEQ ID NO:27;

[0170] D189, E274, and D354 of SEQ ID NO:28; and

[0171] D188, E272 and D352 of SEQ ID NO:29.

[0172] The inventors discovered that the TnpB endonuclease can be unarmed by modifying the DDE motif. Therefore, this disclosure also provides a modified / unarmed TnpB polypeptide comprising a modified DDE motif. In some embodiments, the DDE motif is modified by replacing at least one amino acid in the motif with a neutral or basic amino acid. In some embodiments, at least one amino acid in the motif is replaced with alanine. In some embodiments, the modified TnpB polypeptide contains…

[0173] D186A, E280A and / or D362A compared to SEQ ID NO:1;

[0174] D188A, E272A and / or D368A compared to SEQ ID NO:2;

[0175] D205A, E289A and / or D372A compared to SEQ ID NO:3;

[0176] D184A, E268A and / or D348A compared to SEQ ID NO:4;

[0177] D175A, E260A and / or D339A compared to SEQ ID NO:5;

[0178] D186A, E270A and / or D352A compared to SEQ ID NO:6;

[0179] D181A, E265A and / or D361A compared to SEQ ID NO:7;

[0180] D199A, E293A and / or D383A compared to SEQ ID NO:8;

[0181] D211A, E295A and / or D373A compared to SEQ ID NO:9;

[0182] D185A, E268A and / or D350A compared to SEQ ID NO:10;

[0183] D181A, E265A and / or D345A compared to SEQ ID NO:11;

[0184] D234A, E342A and / or D436A compared to SEQ ID NO:12;

[0185] D184A, E268A and / or D350A compared to SEQ ID NO:13;

[0186] D185A, E269A and / or D351A compared to SEQ ID NO:14;

[0187] D189A, E273A and / or D369A compared to SEQ ID NO:15;

[0188] D190A, E290A and / or D376A compared to SEQ ID NO:16;

[0189] D188A, E272A and / or D368A compared to SEQ ID NO:17;

[0190] D187A, E271A and / or D351A compared to SEQ ID NO:18;

[0191] D188A, E272A and / or D368A compared to SEQ ID NO:19;

[0192] D181A, E265A and / or D345A compared to SEQ ID NO:20;

[0193] D184A, E268A and / or D364A compared to SEQ ID NO:21;

[0194] D183A, E267A and / or D349A compared to SEQ ID NO:22;

[0195] D186A, E270A and / or D351A compared to SEQ ID NO:23;

[0196] D185A, E279A and / or D361A compared to SEQ ID NO:24;

[0197] D188A, E272A and / or D352A compared to SEQ ID NO:25;

[0198] D181A, E265A and / or D348A compared to SEQ ID NO:26;

[0199] D186A, E276A and / or D359A compared to SEQ ID NO:27;

[0200] D189A, E274A and / or D354A compared to SEQ ID NO:28; and

[0201] D188A, E272A and / or D352A compared to SEQ ID NO:29.

[0202] In some embodiments, the modified TnpB polypeptide comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the modified TnpB polypeptide consists of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% of SEQ ID NO:7. In some embodiments, the modified TnpB polypeptide is conserved at the positions corresponding to N31, G179, L267, C332, C335, C351, and C354 of SEQ ID NO:7.

[0203] The inventors further discovered that the TnpB endonuclease can be unarmed by modifying the amino acid corresponding to N31 of SEQ ID NO:7. Therefore, this disclosure also provides a modified / unarmed TnpB polypeptide containing a modification at the N31 position corresponding to SEQ ID NO:7. In some embodiments, the modification is a substitution with alanine.

[0204] In some embodiments, the modified TnpB polypeptide comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the modified TnpB polypeptide consists of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the modified TnpB polypeptide is conserved at the positions corresponding to G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO:7.

[0205] In some implementations, the modified TnpB peptide has the ability to recognize and bind DNA molecules, but loses the ability to cleave DNA molecules such as double-stranded DNA; this is known as a disarmed TnpB peptide.

[0206] 3. Fusion peptides

[0207] This disclosure provides a fusion polypeptide comprising the TnpB polypeptide of this disclosure or a functional fragment thereof, or a modified / unarmed TnpB polypeptide of this disclosure, fused with a fusion partner. The TnpB polypeptide includes a TnpB polypeptide or a functional fragment thereof having RNA-directed endonuclease activity as described above. The modified / unarmed TnpB polypeptide has the ability to recognize and bind DNA molecules, but loses the ability to cleave DNA molecules such as double-stranded DNA.

[0208] In some implementations, the fusion chaperone is a polypeptide that provides indirect transcriptional enhancement activity by acting directly on the target DNA or on polypeptides associated with the target DNA (e.g., histones or other DNA-binding proteins).

[0209] In a further embodiment, the fusion partner is a polypeptide that provides methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristylation activity, or demyristylation activity.

[0210] In further embodiments, the fusion chaperone is a polypeptide that directly provides increased transcription of the target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule / drug-responsive transcription regulator, etc.). In some embodiments, the fusion chaperone is another polypeptide or domain that generates a double-strand break, such as Clo51 or FokI nuclease (Guilinger et al. Nature Biotechnology, volume 32, number 6, June 2014).

[0211] In some implementations, the fusion chaperone is a polypeptide that directs the editing of single or multiple bases in a polynucleotide sequence, such as a site-specific deaminase that can alter the identity of a nucleotide, for example, from CG to TA or from AT to GC (Gaudelli et al., Programmable base editing of AT to GC in genomic DNA without DNA cleavage. Nature (2017); Nishida et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353(6305)(2016); Komor et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533(7603)(2016):420-4).

[0212] The fusion polypeptide may contain, for example, an active (generating double-strand breaks), partially active (nicking enzyme), or inactive (uncleaning) TnpB endonuclease and deaminase (e.g., but not limited to cytidine deaminase, adenine deaminase, APOBEC1, APOBEC3A, BE2, BE3, BE4, ABE, etc.). In some embodiments, the fusion chaperone includes a base editing repair inhibitor and a glycosylation enzyme inhibitor (e.g., a uracil glycosylation enzyme inhibitor (to prevent uracil removal)).

[0213] In some implementations, the fusion partner may be a Cas endonuclease or another TnpB endonuclease as described in this disclosure.

[0214] The TnpB peptide, its functional fragment, or modified / unarmed TnpB disclosed herein can also be fused with a heterologous nuclear localization sequence (NLS). The heterologous NLS described herein can have sufficient strength to drive the TnpB peptide, its functional fragment, modified / unarmed TnpB peptide, or fusion peptide to accumulate in a detectable amount in the eukaryotic nucleus. The NLS can contain one (single) or more (e.g., dual) short sequences (e.g., 2-20 residues) of basic, positively charged residues (e.g., lysine and / or arginine). For example, the NLS can be operatively linked to the N-terminus or C-terminus of the TnpB peptide. Two or more NLS sequences can be linked to the TnpB peptide, for example, at the N-terminus or C-terminus of the TnpB peptide.

[0215] 4. Guide polynucleotides

[0216] Guide polynucleotides enable TnpB peptides to recognize, bind to, and optionally cleave targets. The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combinatorial sequence). Optionally, the guide polynucleotide may contain at least one nucleotide, a phosphodiester bond, or a linker modification, such as, but not limited to, locked nucleic acid (LNA), 5-methyldC, 2,6-diaminopurine, 2'-fluoroA, 2'-fluoroU, 2'-O-methylRNA, a thiophosphate bond, a linker to a cholesterol molecule, a linker to a polyethylene glycol molecule, a linker to a spacer 18 (hexaethylene glycol chain) molecule, or a 5' to 3' covalent linker leading to cyclization. Guide polynucleotides containing only ribonucleic acid are also called "guide RNA" or "gRNA". Guide polynucleotides can be engineered or synthetic. The gRNA of the TnpB peptide is also called "right-element RNA" or "reRNA".

[0217] Guide polynucleotides include chimeric non-naturally occurring guide RNAs, which contain regions that do not coexist in nature (i.e., they are heterologous to each other). For example, a chimeric non-naturally occurring guide RNA contains a targeting region that can hybridize with a nucleotide sequence in a target DNA, linked to a backbone region that can recognize a TnpB polypeptide, wherein the first and second nucleotide sequences are not found linked together in nature. In some embodiments, the targeting region is located at the 3' end of the scaffold.

[0218] The guide polynucleotide of the TnpB peptide is a single guide, and the backbone can be 115-350 nucleotides, for example, at least 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 260, 270, 280, 290, 300, 310, 320, 330, or 340 nucleotides prior to the right-hand (RE) terminus of the derived TnpB. The guide polynucleotide can influence the target recognition, binding, and optionally cleavage of the TnpB peptide when one or more stem structures are removed from the backbone.

[0219] The guide polynucleotide may further include an additional nucleotide sequence at the 5' end of the backbone. In some embodiments, the additional nucleotide sequence may recognize and / or bind to additional nucleases, such as the TnpB polypeptide or Cas nuclease disclosed herein.

[0220] In some implementations, the target region and backbone region are selected from DNA sequences, RNA sequences, and combinations thereof.

[0221] In some implementations, the guide polynucleotide comprises RNA backbone modifications that enhance stability, DNA backbone modifications that enhance stability, and combinations thereof (see Kanasty et al., 2013, Common RNA-backbonemodifications, Nature Materials 12:976-977; US20150082478 published on March 19, 2015 and US20150059010 published on February 26, 2015).

[0222] 5. Polynucleotides and constructs for expressing TnpB peptides or fusion peptides

[0223] The disclosed TnpB endonuclease, its functional fragment, disarmed TnpB polypeptide, and fusion polypeptide can be isolated from natural sources (for TnpB polypeptides) or from recombinant sources, wherein the host cell is genetically modified to express the nucleotide sequence encoding the polypeptide. Alternatively, the TnpB polypeptide and fusion polypeptide can be produced using a cell-free protein expression system or synthesized.

[0224] Therefore, this disclosure also provides an isolated polynucleotide comprising the nucleotide sequence encoding the TnpB polypeptide of this disclosure, a functional fragment thereof, a disarmed TnpB polypeptide, and a fusion polypeptide.

[0225] The TnpB polypeptide, its functional fragments, disarmed TnpB polypeptides, fusion polypeptides, and guide polynucleotides can be expressed in cells. Cells include, but are not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, and plant cells.

[0226] The standard recombinant DNA and molecular cloning techniques used in this paper are well known in the art and are described more fully in Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989). The transformation methods are well known to those skilled in the art and are described below.

[0227] Vectors and constructs, including circular plasmids and linear polynucleotides, are also provided, containing the polynucleotide of interest and other optional components, including adapters, aptamers, and regulatory sequences.

[0228] In some implementations, the vector comprises an expression cassette encoding a TnpB polypeptide and a guide polynucleotide. In some instances, the recognition site and / or target site may be contained within an intron, coding sequence, 5'UTR, 3'UTR, and / or regulatory region.

[0229] In some implementations, the vector contains two expression cassettes encoding a TnpB polypeptide and a guide polynucleotide, respectively.

[0230] In some implementations, the expression of the TnpB peptide and / or guide polynucleotide is driven by a constitutive promoter, an inducible promoter, or a space- and time-specific promoter.

[0231] 6. Gene editing of TnpB peptide

[0232] 6.1. Recombinant gene editing system

[0233] This disclosure provides a recombinant gene editing system comprising a novel TnpB polypeptide of this disclosure that has RNA-guided endonuclease activity.

[0234] In some implementations, the recombinant gene editing system comprises:

[0235] - A TnpB polypeptide or functional fragment thereof of the present disclosure having RNA-directed endonuclease activity, or a polynucleotide comprising a nucleotide sequence encoding a TnpB polypeptide or functional fragment thereof, and

[0236] - Guide polynucleotides, such as guide RNA (gRNA), contain a targeting region capable of hybridizing with a nucleotide sequence of interest and a backbone region capable of binding to a TnpB peptide or a functional fragment thereof, or a polynucleotide containing a nucleotide sequence encoding gRNA.

[0237] In some embodiments, the TnpB polypeptide comprises an N-terminal HTH domain, a central domain, a C-terminal zinc finger domain, and a DDE motif.

[0238] In some embodiments, the TnpB polypeptide is derived from microorganisms selected from Enterococcus faecalis, Aeromonas salmonidae, Clostridium perfringens, Methanococcus martensii, Bacillus cereus, Aeromonas intermedius, Thermobifidafusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc coli PCC7120, Clostridium botulinum type C-Stockholm phage c-st, Methanococcus acetophilus, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Ornithine-lysinic Raoultella, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Airborne Bacillus pallida, and Bacillus thuringiensis.

[0239] In some embodiments, the TnpB polypeptide is encoded by the tnpB gene in an insert sequence (IS) selected from ISEfa4, ISAs26, ISCpe2, ISMma22, ISBce3, ISAeme8, ISTfu1, ISCco1, ISSoc3, ISTel2, ISNsp3, ISCbt1, ISMac7, ISEc46, ISSen6, ISAhl1, ISKpn69, ISDge10, ISKpn85, ISNsp2, ISAba30, ISRor9, ISAam1, ISYmu1, ISCytsp1, ISCvi1, ISCvi2, ISAepa1, and ISBth16.

[0240] In some embodiments, the TnpB polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the TnpB polypeptide is composed of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.

[0241] In some embodiments, the TnpB polypeptide is a functional variant comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the TnpB polypeptide is a functional variant consisting of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. The variants may differ from SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 in that one or more amino acids are substituted, inserted, deleted, and / or added. In some embodiments, the positions of the variants corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO:7 are conserved.

[0242] In some embodiments, the functional fragment comprises at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the functional fragment consists of at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.

[0243] In some embodiments, the TnpB polypeptide fragment recognizes a TAM adjacent to the nucleotide sequence of interest and possesses endonuclease activity. Compared to the ISDra2 TnpB polypeptide, the TnpB polypeptide of this disclosure can recognize shorter TAMs. In some embodiments, the TnpB polypeptide of this disclosure can recognize a TAM consisting of four consecutive nucleotides.

[0244] In some embodiments, the TnpB peptide of this disclosure can recognize the TAM of CCAT, CTAC, TGAC, TGAT, TTAC, TTAG, TTAA, TTAT, ACAT, TTTAT, TTTAA, or TTGAT.

[0245] In some embodiments, the TnpB peptide recognizes the TAM of CCAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 2, 9, 15, 17, or 19.

[0246] In some embodiments, the TnpB peptide recognizes the TAM of CTAC. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:16.

[0247] In some embodiments, the TnpB peptide recognizes the TAM of TGAY, where Y = C or T. In some embodiments, the TnpB peptide comprises amino acids of SEQ ID NO: 7, 10, or 21. In some embodiments, the TnpB peptide recognizes the TAM of TGAC. In some embodiments, the TnpB peptide comprises amino acids of SEQ ID NO: 10 or 21. In some embodiments, the TnpB peptide recognizes the TAM of TGAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 7.

[0248] In some embodiments, the TnpB peptide recognizes the TAM of TTAN, where N is any nucleotide. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, 8, 11, 12, 13, 14, 18, 20, 22, 26, 28, and 29. In some embodiments, the TnpB peptide recognizes the TAM of TTAC. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 5, 11, 12, or 20. In some embodiments, the TnpB peptide recognizes the TAM of TTAG. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 4, 6, or 14. In some embodiments, the TnpB peptide recognizes the TAM of TTAA. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 1, 22, 28, or 29. In some embodiments, the TnpB peptide recognizes the TAM of TTAT. In some embodiments, the TnpB polypeptide comprises an amino acid sequence of SEQ ID NO: 8, 13, 18 or 26.

[0249] In some embodiments, the TnpB peptide recognizes the TAM of TTTAW, where W = A or T. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TnpB peptide recognizes the TAM of TTTAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:23. In some embodiments, the TnpB peptide recognizes the TAM of TTTAA. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:25 or 27.

[0250] In some embodiments, the TnpB peptide recognizes the TAM of TTGAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:24.

[0251] In some embodiments, the TnpB polypeptide or a functional fragment thereof disclosed herein is capable of RNA-directed cleavage in prokaryotic and / or eukaryotic cells, preferably in prokaryotic and eukaryotic cells. In some embodiments, the TnpB polypeptide comprises the amino acid sequence of SEQ ID NO: 7, 18, 21, 23, or 24. In some embodiments, the TnpB polypeptide consists of the amino acid sequence of SEQ ID NO: 7, 18, 21, 23, or 24.

[0252] In some embodiments, the TnpB polypeptide is a functional variant comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:7, 18, 21, 23, or 24. In some embodiments, the TnpB polypeptide is a functional variant composed of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:7, 18, 21, 23, or 24. The variants may differ from SEQ ID NO:7, 18, 21, 23, or 24 in that they involve the substitution, insertion, deletion, and / or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some embodiments, the positions of the variants corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO:7 are conserved.

[0253] In some embodiments, the functional fragment comprises at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 7, 18, 21, 23, or 24. In some embodiments, the functional fragment consists of at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 7, 18, 21, 23, or 24.

[0254] In some implementations, the TnpB contains a DDE motif, corresponding to

[0255] D186, E280, and D362 of SEQ ID NO:1;

[0256] D188, E272 and D368 of SEQ ID NO:2;

[0257] D205, E289, and D372 of SEQ ID NO:3;

[0258] D184, E268, and D348 of SEQ ID NO:4;

[0259] D175, E260, and D339 of SEQ ID NO:5;

[0260] D186, E270, and D352 of SEQ ID NO:6;

[0261] D181, E265, and D361 of SEQ ID NO:7;

[0262] D199, E293, and D383 of SEQ ID NO:8;

[0263] D211, E295, and D373 of SEQ ID NO:9;

[0264] D185, E268, and D350 of SEQ ID NO:10;

[0265] D181, E265, and D345 of SEQ ID NO:11;

[0266] D234, E342, and D436 of SEQ ID NO:12;

[0267] D184, E268, and D350 of SEQ ID NO:13;

[0268] D185, E269, and D351 of SEQ ID NO:14;

[0269] D189, E273, and D369 of SEQ ID NO:15;

[0270] D190, E290, and D376 of SEQ ID NO:16;

[0271] D188, E272, and D368 of SEQ ID NO:17;

[0272] D187, E271, and D351 of SEQ ID NO:18;

[0273] D188, E272, and D368 of SEQ ID NO:19;

[0274] D181, E265, and D345 of SEQ ID NO:20;

[0275] D184, E268, and D364 of SEQ ID NO:21;

[0276] D183, E267, and D349 of SEQ ID NO:22;

[0277] D186, E270, and D351 of SEQ ID NO:23;

[0278] D185, E279, and D361 of SEQ ID NO:24;

[0279] D188, E272 and D352 of SEQ ID NO:25;

[0280] D181, E265, and D348 of SEQ ID NO:26;

[0281] D186, E276, and D359 of SEQ ID NO:27; and

[0282] D189, E274, and D354 of SEQ ID NO:28;

[0283] D188, E272 and D352 of SEQ ID NO:29.

[0284] In some embodiments, the recombinant gene editing system comprises a first polynucleotide and a second polynucleotide, the first polynucleotide comprising a nucleotide sequence encoding a TnpB polypeptide or a functional fragment thereof, and the second polynucleotide comprising a nucleotide sequence encoding a gRNA.

[0285] In some implementations, the recombinant gene editing system further comprises heterologous polynucleotides, such as expression cassettes, transgenes, donor DNA, or polynucleotide modification templates.

[0286] In some embodiments, the backbone of the guide polynucleotide comprises 115-350 nucleotides, for example, at least 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 260, 270, 280, 290, 300, 310, 320, 330, and 340 nucleotides prior to the right-hand (RE) terminus of the derived TnpB. In some embodiments, the backbone is modified by removing part or all of one or more stem structures from the backbone. In some embodiments, part or all of the first stem structure starting from the 3' end is removed.

[0287] In some embodiments, the guide polynucleotide includes an additional nucleotide sequence at the 5' end of the backbone. In some embodiments, the additional nucleotide sequence may recognize and / or bind to additional nucleases, such as the TnpB polypeptide or Cas nuclease disclosed herein.

[0288] In some embodiments, the gRNA further includes one or more additional protein-binding domains. In some embodiments, the system includes one or more additional effector peptides capable of binding to one or more additional protein-binding domains, or a polynucleotide comprising a nucleotide sequence encoding one or more effector peptides to form one or more ribonucleoproteins in tandem.

[0289] 6.2. Compositions, complexes, and isolated cells

[0290] During gene editing, complexes or compositions can be formed. Therefore, this disclosure provides a composition or complex comprising...

[0291] - A recombinant TnpB polypeptide or a functional fragment thereof of the present disclosure having RNA-directed endonuclease activity.

[0292] - Target double-stranded DNA containing the nucleotide sequence of interest and the TAM recognized by the TnpB peptide; and

[0293] - Recombinant guide RNA (gRNA) contains a targeting region capable of hybridizing with a nucleotide sequence of interest and a backbone region capable of binding to a TnpB peptide or a functional fragment thereof.

[0294] This disclosure also provides an isolated cell comprising

[0295] - A recombinant TnpB polypeptide or a functional fragment thereof of the present disclosure having RNA-directed endonuclease activity, or a polynucleotide comprising a nucleotide sequence encoding a TnpB polypeptide or a functional fragment thereof.

[0296] - Target double-stranded DNA containing the nucleotide sequence of interest and the TAM recognized by the TnpB peptide; and

[0297] - Recombinant guide RNA (gRNA) contains a targeting region capable of hybridizing with a nucleotide sequence of interest and a backbone region capable of binding to a TnpB peptide or a functional fragment thereof.

[0298] In some embodiments, the TnpB polypeptide comprises an N-terminal HTH domain, a central domain, a C-terminal zinc finger domain, and a DDE motif.

[0299] In some embodiments, the TnpB polypeptide is derived from microorganisms selected from Enterococcus faecalis, Aeromonas salmonidae, Clostridium perfringens, Methanococcus martensii, Bacillus cereus, Aeromonas intermedius, Thermobifidafusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc coli PCC7120, Clostridium botulinum type C-Stockholm phage c-st, Methanococcus acetophilus, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Ornithine-lysinic Raoultella, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Airborne Bacillus pallida, and Bacillus thuringiensis.

[0300] In some embodiments, the TnpB polypeptide is encoded by the tnpB gene in an insert sequence (IS) selected from ISEfa4, ISAs26, ISCpe2, ISMma22, ISBce3, ISAeme8, ISTfu1, ISCco1, ISSoc3, ISTel2, ISNsp3, ISCbt1, ISMac7, ISEc46, ISSen6, ISAhl1, ISKpn69, ISDge10, ISKpn85, ISNsp2, ISAba30, ISRor9, ISAam1, ISYmu1, ISCytsp1, ISCvi1, ISCvi2, ISAepa1, and ISBth16.

[0301] In some embodiments, the TnpB polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the TnpB polypeptide is composed of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.

[0302] In some embodiments, the TnpB polypeptide is a functional variant comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the TnpB polypeptide is a functional variant consisting of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. The variants may differ from SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 in that one or more amino acids are substituted, inserted, deleted, and / or added. In some embodiments, the positions of the variants corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO:7 are conserved.

[0303] In some embodiments, the functional fragment comprises at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the functional fragment consists of at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.

[0304] In some embodiments, the TnpB polypeptide fragment recognizes a TAM adjacent to the nucleotide sequence of interest and possesses endonuclease activity. Compared to the ISDra2 TnpB polypeptide, the TnpB polypeptide of this disclosure can recognize shorter TAMs. In some embodiments, the TnpB polypeptide of this disclosure can recognize a TAM consisting of four consecutive nucleotides.

[0305] In some embodiments, the TnpB peptide of this disclosure can recognize the TAM of CCAT, CTAC, TGAC, TGAT, TTAC, TTAG, TTAA, TTAT, ACAT, TTTAT, TTTAA, or TTGAT.

[0306] In some embodiments, the TnpB peptide recognizes the TAM of CCAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 2, 9, 15, 17, or 19.

[0307] In some embodiments, the TnpB peptide recognizes the TAM of CTAC. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:16.

[0308] In some embodiments, the TnpB peptide recognizes the TAM of TGAY, where Y = C or T. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 7, 10, or 21. In some embodiments, the TnpB peptide recognizes the TAM of TGAC. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 10 or 21. In some embodiments, the TnpB peptide recognizes the TAM of TGAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 7.

[0309] In some embodiments, the TnpB peptide recognizes the TAM of TTAN, where N is any nucleotide. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, 8, 11, 12, 13, 14, 18, 20, 22, 26, 28, and 29. In some embodiments, the TnpB peptide recognizes the TAM of TTAC. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 5, 11, 12, or 20. In some embodiments, the TnpB peptide recognizes the TAM of TTAG. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 4, 6, or 14. In some embodiments, the TnpB peptide recognizes the TAM of TTAA. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 1, 22, 28, or 29. In some embodiments, the TnpB peptide recognizes the TAM of TTAT. In some embodiments, the TnpB polypeptide comprises an amino acid sequence of SEQ ID NO: 8, 13, 18 or 26.

[0310] In some embodiments, the TnpB peptide recognizes the TAM of TTTAW, where W = A or T. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TnpB peptide recognizes the TAM of TTTAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:23. In some embodiments, the TnpB peptide recognizes the TAM of TTTAA. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:25 or 27.

[0311] In some embodiments, the TnpB peptide recognizes the TAM of TTGAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:24.

[0312] In some embodiments, the TnpB polypeptide or a functional fragment thereof disclosed herein is capable of RNA-directed cleavage in prokaryotic and / or eukaryotic cells, preferably in prokaryotic and eukaryotic cells. In some embodiments, the TnpB polypeptide comprises the amino acid sequence of SEQ ID NO: 7, 18, 21, 23, or 24. In some embodiments, the TnpB polypeptide consists of the amino acid sequence of SEQ ID NO: 7, 18, 21, 23, or 24.

[0313] In some embodiments, the TnpB polypeptide is a functional variant comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:7, 18, 21, 23, or 24. In some embodiments, the TnpB polypeptide is a functional variant composed of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:7, 18, 21, 23, or 24. The variants may differ from SEQ ID NO:7, 18, 21, 23, or 24 in that they involve the substitution, insertion, deletion, and / or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some embodiments, the positions of the variants corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO:7 are conserved.

[0314] In some embodiments, the functional fragment comprises at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 7, 18, 21, 23, or 24. In some embodiments, the functional fragment consists of at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 7, 18, 21, 23, or 24.

[0315] In some implementations, the TnpB contains a DDE motif, corresponding to

[0316] D186, E280, and D362 of SEQ ID NO:1;

[0317] D188, E272 and D368 of SEQ ID NO:2;

[0318] D205, E289, and D372 of SEQ ID NO:3;

[0319] D184, E268, and D348 of SEQ ID NO:4;

[0320] D175, E260, and D339 of SEQ ID NO:5;

[0321] D186, E270, and D352 of SEQ ID NO:6;

[0322] D181, E265, and D361 of SEQ ID NO:7;

[0323] D199, E293, and D383 of SEQ ID NO:8;

[0324] D211, E295, and D373 of SEQ ID NO:9;

[0325] D185, E268, and D350 of SEQ ID NO:10;

[0326] D181, E265, and D345 of SEQ ID NO:11;

[0327] D234, E342, and D436 of SEQ ID NO:12;

[0328] D184, E268, and D350 of SEQ ID NO:13;

[0329] D185, E269, and D351 of SEQ ID NO:14;

[0330] D189, E273, and D369 of SEQ ID NO:15;

[0331] D190, E290, and D376 of SEQ ID NO:16;

[0332] D188, E272, and D368 of SEQ ID NO:17;

[0333] D187, E271, and D351 of SEQ ID NO:18;

[0334] D188, E272, and D368 of SEQ ID NO:19;

[0335] D181, E265, and D345 of SEQ ID NO:20;

[0336] D184, E268, and D364 of SEQ ID NO:21;

[0337] D183, E267, and D349 of SEQ ID NO:22;

[0338] D186, E270, and D351 of SEQ ID NO:23;

[0339] D185, E279, and D361 of SEQ ID NO:24;

[0340] D188, E272 and D352 of SEQ ID NO:25;

[0341] D181, E265, and D348 of SEQ ID NO:26;

[0342] D186, E276, and D359 of SEQ ID NO:27;

[0343] D189, E274, and D354 of SEQ ID NO:28;

[0344] D188, E272 and D352 of SEQ ID NO:29.

[0345] In some implementations, the recombinant gene editing system comprises a first polynucleotide and a second polynucleotide, the first polynucleotide comprising a nucleotide sequence encoding a TnpB polypeptide or a functional fragment thereof, and the second polynucleotide comprising a nucleotide sequence encoding a gRNA.

[0346] In some embodiments, the composition further comprises heterologous polynucleotides, such as expression cassettes, transgenes, donor DNA, or polynucleotide modified templates.

[0347] In some embodiments, the backbone of the guide polynucleotide comprises 115-350 nucleotides, for example, at least 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 260, 270, 280, 290, 300, 310, 320, 330, and 340 nucleotides prior to the right-hand (RE) terminus of the derived TnpB. In some embodiments, the backbone is modified by removing part or all of one or more stem structures from the backbone. In some embodiments, part or all of the first stem structure starting from the 3' end is removed.

[0348] In some embodiments, the guide polynucleotide includes an additional nucleotide sequence at the 5' end of the backbone. In some embodiments, the additional nucleotide sequence may recognize and / or bind to additional nucleases, such as the TnpB polypeptide or Cas nuclease disclosed herein.

[0349] In some embodiments, the gRNA further comprises one or more additional protein-binding domains. In some embodiments, the composition for isolating cells comprises one or more additional effector peptides capable of binding to one or more additional protein-binding domains, or comprises a polynucleotide comprising a nucleotide sequence encoding one or more effector peptides to tandemly form one or more ribonucleoproteins.

[0350] 6.3. Disarmament System

[0351] This disclosure provides a recombination system comprising:

[0352] - The modified TnpB polypeptide disclosed herein contains a modification, or a functional fragment thereof, in the DDE motif compared to the parental TnpB polypeptide, or a polynucleotide containing a nucleotide sequence encoding the modified TnpB polypeptide or a functional fragment thereof, wherein the parental polypeptide possesses RNA-directed endonuclease activity, and the modified TnpB polypeptide loses its activity in cleaving double-stranded DNA, and

[0353] - Guide RNA (gRNA) contains a target region capable of hybridizing to a nucleotide sequence of interest and a backbone region capable of binding to a TnpB polypeptide or a functional fragment thereof, or a polynucleotide containing a nucleotide sequence encoding gRNA.

[0354] In some embodiments, the TnpB polypeptide comprises an N-terminal HTH domain, a central domain, a C-terminal zinc finger domain, and a DDE motif.

[0355] In some embodiments, the DDE motif is modified by replacing at least one amino acid in the motif with a neutral or basic amino acid. In some embodiments, at least one amino acid in the motif is replaced with alanine. In some embodiments, the modified TnpB polypeptide contains...

[0356] D186A, E280A and / or D362A compared to SEQ ID NO:1;

[0357] D188A, E272A and / or D368A compared to SEQ ID NO:2;

[0358] D205A, E289A and / or D372A compared to SEQ ID NO:3;

[0359] D184A, E268A and / or D348A compared to SEQ ID NO:4;

[0360] D175A, E260A and / or D339A compared to SEQ ID NO:5;

[0361] D186A, E270A and / or D352A compared to SEQ ID NO:6;

[0362] D181A, E265A and / or D361A compared to SEQ ID NO:7;

[0363] D199A, E293A and / or D383A compared to SEQ ID NO:8;

[0364] D211A, E295A and / or D373A compared to SEQ ID NO:9;

[0365] D185A, E268A and / or D350A compared to SEQ ID NO:10;

[0366] D181A, E265A and / or D345A compared to SEQ ID NO:11;

[0367] D234A, E342A and / or D436A compared to SEQ ID NO:12;

[0368] D184A, E268A and / or D350A compared to SEQ ID NO:13;

[0369] D185A, E269A and / or D351A compared to SEQ ID NO:14;

[0370] D189A, E273A and / or D369A compared to SEQ ID NO:15;

[0371] D190A, E290A and / or D376A compared to SEQ ID NO:16;

[0372] D188A, E272A and / or D368A compared to SEQ ID NO:17;

[0373] D187A, E271A and / or D351A compared to SEQ ID NO:18;

[0374] D188A, E272A and / or D368A compared to SEQ ID NO:19;

[0375] D181A, E265A and / or D345A compared to SEQ ID NO:20;

[0376] D184A, E268A and / or D364A compared to SEQ ID NO:21;

[0377] D183A, E267A and / or D349A compared to SEQ ID NO:22;

[0378] D186A, E270A and / or D351A compared to SEQ ID NO:23;

[0379] D185A, E279A and / or D361A compared to SEQ ID NO:24;

[0380] D188A, E272A and / or D352A compared to SEQ ID NO:25;

[0381] D181A, E265A and / or D348A compared to SEQ ID NO:26;

[0382] D186A, E276A and / or D359A compared to SEQ ID NO:27;

[0383] D189A, E274A and / or D354A compared to SEQ ID NO:28; and

[0384] D188A, E272A and / or D352A compared to SEQ ID NO:29.

[0385] In some embodiments, the modified TnpB polypeptide comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the modified TnpB polypeptide consists of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% of SEQ ID NO:7. In some embodiments, the modified TnpB polypeptide is conserved at the positions corresponding to N31, G179, L267, C332, C335, C351, and C354 of SEQ ID NO:7.

[0386] In some embodiments, the modified TnpB polypeptide contains a modification at position N31 corresponding to SEQ ID NO:7. In some embodiments, the modification is a substitution of alanine.

[0387] In some embodiments, the modified TnpB polypeptide comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the modified TnpB polypeptide consists of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the modified TnpB polypeptide is conserved at the positions corresponding to G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO:7.

[0388] In some embodiments, the modified TnpB peptide has the ability to recognize and bind DNA molecules, but loses the ability to cleave DNA molecules such as double-stranded DNA, i.e., it is a disarmed TnpB peptide.

[0389] In some embodiments, the backbone of the guide polynucleotide comprises 115-350 nucleotides, for example, at least 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 260, 270, 280, 290, 300, 310, 320, 330, and 340 nucleotides prior to the right-hand (RE) terminus of the derived TnpB. In some embodiments, the backbone is modified by removing part or all of one or more stem structures from the backbone. In some embodiments, part or all of the first stem structure starting from the 3' end is removed.

[0390] In some embodiments, the guide polynucleotide includes an additional nucleotide sequence at the 5' end of the backbone. In some embodiments, said additional nucleotide sequence may recognize and / or bind to additional nucleases, such as the TnpB polypeptide or Cas nuclease disclosed herein.

[0391] In some embodiments, the gRNA further includes one or more additional protein-binding domains. In some embodiments, the system includes one or more additional effector peptides capable of binding to the one or more additional protein-binding domains, or a polynucleotide comprising a nucleotide sequence encoding one or more effector peptides to form one or more ribonucleoproteins in tandem.

[0392] 6.4. Fusion System

[0393] This disclosure provides a gene editing system comprising:

[0394] - The fusion polypeptide disclosed herein, for example, comprises a TnpB polypeptide or a functional fragment thereof having RNA-directed endonuclease activity, or a modified TnpB polypeptide fused to a fusion partner, or a polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide or a functional fragment thereof, and

[0395] - Guide polynucleotides, such as guide RNA (gRNA), contain a targeting region capable of hybridizing with a nucleotide sequence of interest and a backbone region capable of binding to a TnpB peptide or a functional fragment thereof, or a polynucleotide containing a nucleotide sequence encoding gRNA.

[0396] In some embodiments, the TnpB polypeptide comprises an N-terminal HTH domain, a central domain, a C-terminal zinc finger domain, and a DDE motif.

[0397] In some embodiments, the TnpB polypeptide is derived from microorganisms selected from Enterococcus faecalis, Aeromonas salmonidae, Clostridium perfringens, Methanococcus martensii, Bacillus cereus, Aeromonas intermedius, Thermobifidafusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc coli PCC7120, Clostridium botulinum type C-Stockholm phage c-st, Methanococcus acetophilus, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Ornithine-lysinic Raoultella, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Airborne Bacillus pallida, and Bacillus thuringiensis.

[0398] In some embodiments, the TnpB polypeptide is encoded by the tnpB gene in an insert sequence (IS) selected from ISEfa4, ISAs26, ISCpe2, ISMma22, ISBce3, ISAeme8, ISTfu1, ISCco1, ISSoc3, ISTel2, ISNsp3, ISCbt1, ISMac7, ISEc46, ISSen6, ISAhl1, ISKpn69, ISDge10, ISKpn85, ISNsp2, ISAba30, ISRor9, ISAam1, ISYmu1, ISCytsp1, ISCvi1, ISCvi2, ISAepa1, and ISBth16.

[0399] In some embodiments, the TnpB polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the TnpB polypeptide is composed of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.

[0400] In some embodiments, the TnpB polypeptide is a functional variant comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the TnpB polypeptide is a functional variant consisting of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. The variants may differ from SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 in that one or more amino acids are substituted, inserted, deleted, and / or added. In some embodiments, the positions of the variants corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO:7 are conserved.

[0401] In some embodiments, the functional fragment comprises at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the functional fragment consists of at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.

[0402] In some embodiments, the TnpB polypeptide fragment recognizes a TAM adjacent to the nucleotide sequence of interest and possesses endonuclease activity. Compared to the ISDra2 TnpB polypeptide, the TnpB polypeptide of this disclosure can recognize shorter TAMs. In some embodiments, the TnpB polypeptide of this disclosure can recognize a TAM consisting of four consecutive nucleotides.

[0403] In some embodiments, the TnpB peptide of this disclosure can recognize the TAM of CCAT, CTAC, TGAC, TGAT, TTAC, TTAG, TTAA, TTAT, ACAT, TTTAT, TTTAA, or TTGAT.

[0404] In some embodiments, the TnpB peptide recognizes the TAM of CCAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 2, 9, 15, 17, or 19.

[0405] In some embodiments, the TnpB peptide recognizes the TAM of CTAC. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:16.

[0406] In some embodiments, the TnpB peptide recognizes the TAM of TGAY, where Y = C or T. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 7, 10, or 21. In some embodiments, the TnpB peptide recognizes the TAM of TGAC. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 10 or 21. In some embodiments, the TnpB peptide recognizes the TAM of TGAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 7.

[0407] In some embodiments, the TnpB peptide recognizes the TAM of TTAN, where N is any nucleotide. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, 8, 11, 12, 13, 14, 18, 20, 22, 26, 28, and 29. In some embodiments, the TnpB peptide recognizes the TAM of TTAC. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 5, 11, 12, or 20. In some embodiments, the TnpB peptide recognizes the TAM of TTAG. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 4, 6, or 14. In some embodiments, the TnpB peptide recognizes the TAM of TTAA. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO: 1, 22, 28, or 29. In some embodiments, the TnpB peptide recognizes the TAM of TTAT. In some embodiments, the TnpB polypeptide comprises an amino acid sequence of SEQ ID NO: 8, 13, 18 or 26.

[0408] In some embodiments, the TnpB peptide recognizes the TAM of TTTAW, where W = A or T. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TnpB peptide recognizes the TAM of TTTAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:23. In some embodiments, the TnpB peptide recognizes the TAM of TTTAA. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:25 or 27.

[0409] In some embodiments, the TnpB peptide recognizes the TAM of TTGAT. In some embodiments, the TnpB peptide comprises the amino acid sequence of SEQ ID NO:24.

[0410] In some embodiments, the TnpB polypeptide or a functional fragment thereof disclosed herein is capable of RNA-directed cleavage in prokaryotic and / or eukaryotic cells, preferably in prokaryotic and eukaryotic cells. In some embodiments, the TnpB polypeptide comprises the amino acid sequence of SEQ ID NO: 7, 18, 21, 23, or 24. In some embodiments, the TnpB polypeptide consists of the amino acid sequence of SEQ ID NO: 7, 18, 21, 23, or 24.

[0411] In some embodiments, the TnpB polypeptide is a functional variant comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:7, 18, 21, 23, or 24. In some embodiments, the TnpB polypeptide is a functional variant composed of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:7, 18, 21, 23, or 24. The variants may differ from SEQ ID NO:7, 18, 21, 23, or 24 in that they involve the substitution, insertion, deletion, and / or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some embodiments, the positions of the variants corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO:7 are conserved.

[0412] In some embodiments, the functional fragment comprises at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 7, 18, 21, 23, or 24. In some embodiments, the functional fragment consists of at least 100, 150, 200, 225, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 consecutive amino acids selected from SEQ ID NO: 7, 18, 21, 23, or 24.

[0413] In some implementations, the TnpB contains a DDE motif, corresponding to

[0414] D186, E280, and D362 of SEQ ID NO:1;

[0415] D188, E272 and D368 of SEQ ID NO:2;

[0416] D205, E289, and D372 of SEQ ID NO:3;

[0417] D184, E268, and D348 of SEQ ID NO:4;

[0418] D175, E260, and D339 of SEQ ID NO:5;

[0419] D186, E270, and D352 of SEQ ID NO:6;

[0420] D181, E265, and D361 of SEQ ID NO:7;

[0421] D199, E293, and D383 of SEQ ID NO:8;

[0422] D211, E295, and D373 of SEQ ID NO:9;

[0423] D185, E268, and D350 of SEQ ID NO:10;

[0424] D181, E265, and D345 of SEQ ID NO:11;

[0425] D234, E342, and D436 of SEQ ID NO:12;

[0426] D184, E268, and D350 of SEQ ID NO:13;

[0427] D185, E269, and D351 of SEQ ID NO:14;

[0428] D189, E273, and D369 of SEQ ID NO:15;

[0429] D190, E290, and D376 of SEQ ID NO:16;

[0430] D188, E272, and D368 of SEQ ID NO:17;

[0431] D187, E271, and D351 of SEQ ID NO:18;

[0432] D188, E272, and D368 of SEQ ID NO:19;

[0433] D181, E265, and D345 of SEQ ID NO:20;

[0434] D184, E268, and D364 of SEQ ID NO:21;

[0435] D183, E267, and D349 of SEQ ID NO:22;

[0436] D186, E270, and D351 of SEQ ID NO:23;

[0437] D185, E279, and D361 of SEQ ID NO:24;

[0438] D188, E272 and D352 of SEQ ID NO:25;

[0439] D181, E265, and D348 of SEQ ID NO:26;

[0440] D186, E276, and D359 of SEQ ID NO:27;

[0441] D189, E274, and D354 of SEQ ID NO:28; and

[0442] D188, E272 and D352 of SEQ ID NO:29.

[0443] In some implementations, the recombinant gene editing system comprises a first polynucleotide and a second polynucleotide, the first polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide and the second polynucleotide comprising a nucleotide sequence encoding a gRNA.

[0444] In some implementations, the recombinant gene editing system further includes heterologous polynucleotides, such as expression cassettes, transgenes, donor DNA, or polynucleotide modification templates.

[0445] In some embodiments, the backbone of the guide polynucleotide comprises 115-350 nucleotides, for example, at least 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 260, 270, 280, 290, 300, 310, 320, 330, and 340 nucleotides prior to the right-hand (RE) terminus of the derived TnpB. In some embodiments, the backbone is modified by removing part or all of one or more stem structures from the backbone. In some embodiments, part or all of the first stem structure starting from the 3' end is removed.

[0446] In some embodiments, the guide polynucleotide includes an additional nucleotide sequence at the 5' end of the backbone. In some embodiments, the additional nucleotide sequence may recognize and / or bind to additional nucleases, such as the TnpB polypeptide or Cas nuclease disclosed herein.

[0447] In some implementations, the modified TnpB peptide contains modifications in the DDE motif compared to the parental TnpB peptide, wherein the parental peptide contains an N-terminal HTH domain, a central domain, a C-terminal zinc finger domain, and the DDE motif.

[0448] In some embodiments, the DDE motif is modified by replacing at least one amino acid in the motif with a neutral or basic amino acid. In some embodiments, at least one amino acid in the motif is replaced with alanine. In some embodiments, the modified TnpB polypeptide contains...

[0449] D186A, E280A and / or D362A compared to SEQ ID NO:1;

[0450] D188A, E272A and / or D368A compared to SEQ ID NO:2;

[0451] D205A, E289A and / or D372A compared to SEQ ID NO:3;

[0452] D184A, E268A and / or D348A compared to SEQ ID NO:4;

[0453] D175A, E260A and / or D339A compared to SEQ ID NO:5;

[0454] D186A, E270A and / or D352A compared to SEQ ID NO:6;

[0455] D181A, E265A and / or D361A compared to SEQ ID NO:7;

[0456] D199A, E293A and / or D383A compared to SEQ ID NO:8;

[0457] D211A, E295A and / or D373A compared to SEQ ID NO:9;

[0458] D185A, E268A and / or D350A compared to SEQ ID NO:10;

[0459] D181A, E265A and / or D345A compared to SEQ ID NO:11;

[0460] D234A, E342A and / or D436A compared to SEQ ID NO:12;

[0461] D184A, E268A and / or D350A compared to SEQ ID NO:13;

[0462] D185A, E269A and / or D351A compared to SEQ ID NO:14;

[0463] D189A, E273A and / or D369A compared to SEQ ID NO:15;

[0464] D190A, E290A and / or D376A compared to SEQ ID NO:16;

[0465] D188A, E272A and / or D368A compared to SEQ ID NO:17;

[0466] D187A, E271A and / or D351A compared to SEQ ID NO:18;

[0467] D188A, E272A and / or D368A compared to SEQ ID NO:19;

[0468] D181A, E265A and / or D345A compared to SEQ ID NO:20;

[0469] D184A, E268A and / or D364A compared to SEQ ID NO:21;

[0470] D183A, E267A and / or D349A compared to SEQ ID NO:22;

[0471] D186A, E270A and / or D351A compared to SEQ ID NO:23;

[0472] D185A, E279A and / or D361A compared to SEQ ID NO:24;

[0473] D188A, E272A and / or D352A compared to SEQ ID NO:25;

[0474] D181A, E265A and / or D348A compared to SEQ ID NO:26;

[0475] D186A, E276A and / or D359A compared to SEQ ID NO:27;

[0476] D189A, E274A and / or D354A compared to SEQ ID NO:28; and

[0477] D188A, E272A and / or D352A compared to SEQ ID NO:29.

[0478] In some embodiments, the modified TnpB polypeptide comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the modified TnpB polypeptide consists of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% of SEQ ID NO:7. In some embodiments, the modified TnpB polypeptide is conserved at the positions corresponding to N31, G179, L267, C332, C335, C351, and C354 of SEQ ID NO:7.

[0479] In some embodiments, the modified TnpB polypeptide contains a modification at position N31 corresponding to SEQ ID NO:7. In some embodiments, the modification is a substitution of alanine.

[0480] In some embodiments, the modified TnpB polypeptide comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the modified TnpB polypeptide consists of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In some embodiments, the modified TnpB polypeptide is conserved at the positions corresponding to G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO:7.

[0481] In some implementations, the modified TnpB peptide has the ability to recognize and bind DNA molecules, but loses the ability to cleave DNA molecules such as double-stranded DNA; this is known as a disarmed TnpB peptide.

[0482] In some implementations, the fusion chaperone is a polypeptide that provides indirect transcriptional enhancement activity by acting directly on the target DNA or on polypeptides associated with the target DNA (e.g., histones or other DNA-binding proteins).

[0483] In a further embodiment, the fusion partner is a polypeptide that provides methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristylation activity, or demyristylation activity.

[0484] In further embodiments, the fusion chaperone is a polypeptide that directly provides increased transcription of the target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule / drug-responsive transcription regulator, etc.). In some embodiments, the fusion chaperone is another polypeptide or domain that generates a double-strand break, such as Clo51 or FokI nuclease (Guilinger et al. Nature Biotechnology, volume 32, number 6, June 2014).

[0485] In some implementations, the fusion chaperone is a polypeptide that directs the editing of single or multiple bases in a polynucleotide sequence, such as a site-specific deaminase that can alter the identity of a nucleotide, for example, from CG to TA or from AT to GC (Gaudelli et al., Programmable base editing of AT to GC in genomic DNA without DNA cleavage. Nature (2017); Nishida et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353(6305)(2016); Komor et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533(7603)(2016):420-4).

[0486] In some embodiments, the fusion chaperone is an active (double-strand break generated), partially active (nicking enzyme), or inactive (unable to cleave) TnpB endonuclease and deaminase (e.g., but not limited to cytidine deaminase, adenine deaminase, APOBEC1, APOBEC3A, BE2, BE3, BE4, ABE, etc.). In some embodiments, the fusion chaperone includes base editing repair inhibitors and glycosylation enzyme inhibitors (e.g., uracil glycosylation enzyme inhibitors (to prevent uracil removal)).

[0487] In some implementations, the fusion partner is a Cas endonuclease or another TnpB endonuclease as described in this disclosure.

[0488] In some embodiments, the fusion partner is a heterologous NLS. In some embodiments, the NLS is operatively linked to the N-terminus or C-terminus of a TnpB peptide, a functional fragment thereof, or a modified TnpB peptide. In some embodiments, the fusion peptide comprises two or more NLS sequences linked to the TnpB peptide, a functional fragment thereof, or a modified TnpB peptide, for example, at its N- and C-termini.

[0489] In some embodiments, the gRNA further includes one or more additional protein-binding domains. In some embodiments, the system includes one or more additional effector peptides capable of binding to one or more additional protein-binding domains, or a polynucleotide comprising a nucleotide sequence encoding one or more effector peptides to form one or more ribonucleoproteins in tandem.

[0490] 7. Gene Editing Methods

[0491] This disclosure provides a method for introducing a double-strand break into a polynucleotide of interest, comprising the step of contacting the polynucleotide with a recombinant gene editing system of this disclosure that targets a nucleotide sequence in the polynucleotide.

[0492] This disclosure provides a method for modifying a genomic sequence in a cell, including the step of introducing a recombinant gene editing system or fusion system of this disclosure that targets the genomic sequence in the cell into the cell.

[0493] This disclosure provides a method for modifying a genome sequence in a cell, comprising the step of introducing a disarmed system of this disclosure and a gene editing system targeting a genome sequence into the cell, wherein the nucleotide sequence targeted by the disarmed system is close to the genome sequence.

[0494] Methods for introducing polynucleotides or peptides or polynucleotide-protein complexes into cells or organisms are known in the art, including but not limited to microinjection, electroporation, stable transformation methods, transient transformation methods, ballistic particle acceleration (particle bombardment), whisker-mediated transformation, Agrobacterium-mediated transformation, direct gene transfer, virus-mediated introduction, transfection, transduction, cell-penetrating peptides, direct protein delivery mediated by mesoporous silica nanoparticles (MSN), topical application, sexual hybridization, sexual reproduction, and any combination thereof.

[0495] Adeno-associated virus (AAV) is a widely used vector for delivering heterologous polynucleotides. However, due to the small size of the AAV genome (approximately 4.7 kb) and the large size of the Cas peptide (over 1000 amino acids), delivery of Cas systems using recombinant AAV (rAAV) is limited, and it is generally impossible to deliver a single vector-encoded Cas-fusion system (fusion product and gRNA). Compared to Cas systems, gene editing systems containing both the TnpB peptide and the TnpB fusion product can be delivered in a single rAAV due to the much smaller size of the TnpB peptide.

[0496] Therefore, this disclosure provides a recombinant adeno-associated virus (rAAV) whose genome includes a first expression cassette encoding the TnpB polypeptide, a modified TnpB polypeptide, or a fusion polypeptide of this disclosure. In some embodiments, the first expression cassette includes a promoter and a terminator.

[0497] In some embodiments, the genome includes a second expression cassette encoding a guide RNA (gRNA) comprising a targeting region capable of hybridizing to a nucleotide sequence of interest and a backbone region capable of binding to a TnpB peptide or a functional fragment thereof. In some embodiments, the second expression cassette includes a promoter and a terminator.

[0498] In some implementations, the first expression cassette contains fewer than about 4,700 nucleotides, fewer than about 4,600 nucleotides, fewer than about 4,500 nucleotides, fewer than about 4,400 nucleotides, fewer than about 4,300 nucleotides, fewer than about 4,200 nucleotides, fewer than about 4,100 nucleotides, fewer than about 4,000 nucleotides, fewer than about 3,900 nucleotides, fewer than about 3,800 nucleotides, fewer than about 3,700 nucleotides, fewer than about 3,600 nucleotides, fewer than about 3,500 nucleotides, fewer than about 3,400 nucleotides, fewer than about 3,300 nucleotides, fewer than about 3,200 nucleotides, fewer than about 3,100 nucleotides, fewer than about 3,000 nucleotides, fewer than about 2,900 nucleotides, fewer than about 2,800 nucleotides, fewer than about 2,700 nucleotides, fewer than about 2,600 nucleotides, or fewer than about 2,500 nucleotides.

[0499] In some implementations, the genome contains about 4,500 to about 4,700 nucleotides.

[0500] 8. A method for screening TnpB peptides with RNA-directed endonuclease activity.

[0501] This disclosure provides a method for screening the activity of TnpB peptides in cleaving double-stranded DNA, comprising the following steps:

[0502] - Provides candidate TnpB peptides from microorganisms;

[0503] - Provide a gRNA comprising a target region and a backbone region, wherein the backbone region comprises at least 100 nucleotides preceding the 3' end of an IS, and the IS naturally comprises a nucleotide sequence encoding a TnpB polypeptide;

[0504] - Provide target DNA containing a nucleotide sequence that hybridizes to the nucleotide sequence of the target region and a TAM recognized by the TnpB peptide, wherein the TAM consists of four or five consecutive nucleotides adjacent to the 5' end of IS.

[0505] - Enables the TnpB peptide to contact gRNA and target DNA; and

[0506] - Detect cuts on the target DNA.

[0507] In some embodiments, the backbone region comprises at least 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more nucleotides preceding the 3' end of the IS. In some embodiments, the backbone region comprises 100-350, 125-325, 150-300, 175-275, 200-250, 150-225, 175-225, 150-200, 175-225 or 175-200 nucleotides preceding the 3' end of the IS.

[0508] In some embodiments, the TnpB polypeptide comprises an N-terminal HTH domain, a central domain, a C-terminal zinc finger domain, and a DDE motif.

[0509] The TnpB polypeptide can be provided as a separate polypeptide or through the expression of a polynucleotide encoding the polypeptide. In some embodiments, the TnpB polypeptide is provided by a first polynucleotide, preferably DNA, containing a first nucleotide sequence encoding the TnpB polypeptide.

[0510] gRNA can be provided as a separate RNA molecule or through transcription of a DNA molecule encoding the gRNA. In some embodiments, the gRNA is provided as a second polynucleotide, preferably DNA, containing a second nucleotide sequence encoding the gRNA.

[0511] The first and second polynucleotides may be provided in a single vector or in different vectors. In some embodiments, the first and second polynucleotides are provided in a first vector, such as a first plasmid. In some embodiments, the first nucleotide sequence is operatively linked to a first promoter. In some embodiments, the second nucleotide sequence is operatively linked to a second promoter.

[0512] In some implementations, the target DNA is provided in a second plasmid.

[0513] In some implementations, contacting the TnpB peptide with gRNA and target DNA involves introducing first and second plasmids into a host cell containing the target DNA. Example

[0514] Example 1. Screening for TnpB peptides with endonuclease activity

[0515] This embodiment was conducted to screen for TnpB peptides with endonuclease activity.

[0516] A series of plasmid pairs were constructed (a test plasmid containing the nucleotide sequence encoding the TnpB polypeptide, the gRNA (the target sequence of SEQ ID NO:240 and the associated reRNA backbone), and the chloramphenicol (Cm) resistance gene, and a reporter plasmid containing the target sequence of SEQ ID NO:240, the TAM, and the kanamycin resistance gene).

[0517] In short, for the test plasmid, the TnpB gene and gRNA coding sequence (backbone + target region) were synthesized by Tsingke (Beijing, China) and cloned into the pBAD backbone via Gibson assembly. The TnpB gene was driven by the J23108 promoter, and the gRNA coding sequence was driven by the J23119 promoter (see Leeny et al., 2016, Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems, Molecular Cell, 62, 1-11).

[0518] Order a reporter plasmid (Kan+) carrying oligonucleotides from Tsingke (Beijing, China). This plasmid contains the target nucleotide sequence and associated TAM, with EcoRI and XhoI restriction sites flanking it. In short, digest the oligonucleotides and pCB457 plasmid with EcoRI and BamHI at 37°C for 1 hour. Following the manufacturer's instructions, isolate and ligate the digestion products using T4 ligase.

[0519] Taking the spectrum of the ISTfu1 TnpB plasmid pair as an example, such as Figure 1 As shown.

[0520] Each plasmid pair was transformed into E. coli (E. coli) BW25141 cells by electroporation to test the endonuclease activity of the TnpB peptide (test group), and plasmid pairs from which the gRNA coding sequence was removed were used as negative controls.

[0521] Specifically, the test plasmid and target plasmid were electroporated into *E. coli* (NEB 10β, C3020K) using a BIO-RAD machine (Gene Pulser Xcell) at a program of 1.8 kV, 25 μF, and 200 phm. After electroporation, 900 μl of SOC medium was added, followed by incubation at 37°C for 1 hour.

[0522] The mixture was then divided into four portions, serially diluted (10×), and inoculated onto different LB plates (Cm+ / Kan+, Cm+, Kan+, and standard) (50 mg / L for each antibiotic). After incubation at 37°C for 12 hours, the plates were photographed.

[0523] Count the viable colonies and calculate the exhaustion rate (exhaustion rate = number of colonies in the TnpB & reRNA group at the minimum dilution / number of colonies in the TnpB group alone at the minimum dilution).

[0524] Plain plates, K+ plates, and Cm+ plates are used to demonstrate the efficiency of electroporation. Generally, the negative control shows similar colony counts on these three plates compared to the test group.

[0525] On K+Cm+ plates, colonies appeared in the negative control and the test group encoding the TnpB polypeptide without endonuclease activity, while no colonies appeared in the test group encoding the TnpB polypeptide with endonuclease activity.

[0526] A total of 81 TnpB peptides were tested, and the results showed that the TnpB peptide of SEQ ID NO:1-29 exhibited endonuclease activity (see...). Figure 2 ), which cleaves target sites in an RNA-guided manner, exhibiting different depletion ratios (see... Figure 3 ), while the 52 TnpB peptides encoded by SEQ ID NO:59-110 did not show endonuclease activity (data not shown).

[0527] Table 1 shows the TnpB polypeptide and gRNA with RNA-directed endonuclease activity and their TAMs.

[0528] Table 1. TnpB peptides with desired activity

[0529]

[0530]

[0531] Example 2. Characterization of TnpB peptide

[0532] 2.1. Sequence Analysis

[0533] The amino acid sequence of the TnpB peptide exhibiting endonuclease activity was aligned (Clustal Omega). The results showed that the DDE motif is conserved in the TnpB peptide (see [link to original text]). Figure 4 and 5 Although the DDE motif in ISBce3 TnpB and ISCbt1TnpB does not perfectly match other motifs, it is indeed present and indicated in the amino acid sequence (see [link]). Figure 5 It is noteworthy that TnpB peptides with RNA-directed endonuclease activity are generally conserved at the positions corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354, and D361 in SEQ ID NO:7, except for ISCbt1 (SEQ ID NO:12), which differs from other peptides at the positions corresponding to L267, C332, C335, C351, and C354 in SEQ ID NO:7.

[0534] For clarity, the comparison between the 25 newly identified TnpB peptides and the ISDra2 TnpB peptide is as follows: Figure 12 As shown in Figure A.

[0535] 2.2. DDE motifs are crucial for endonuclease activity.

[0536] As described in Example 1, the TnpB polypeptide variant of SEQ ID NO:1-22 was tested, wherein the first D residue of the DDE motif was replaced by A. The results showed that the variant essentially lost its endonuclease activity (see Example 1). Figure 2 and 3 This indicates that the DDE motif is crucial for endonuclease activity, and that disarmed TnpB peptides (dTnpB) can be prepared by introducing mutations into the DDE motif.

[0537] 2.3. Conserved amino acid residues are crucial for endonuclease activity.

[0538] As described in Example 1, variants of the TnpB peptides ISTfu1 (SEQ ID NO:7), ISDge10 (SEQ ID NO:18), ISAba30 (SEQ ID NO:21), and ISDra2 were tested, wherein the amino acid corresponding to N31 of SEQ ID NO:7 was substituted with A. The results showed that the variants essentially lost their endonuclease activity (see Example 1). Figure 12 B) indicates that conserved amino acid residues, such as the amino acid residue corresponding to N31 of SEQ ID NO:7, are crucial for endonuclease activity, and that disarmed TnpB peptides (dTnpB) can be prepared by introducing mutations into the conserved amino acids between TnpB peptides.

[0539] Example 3. RNA-guided cleavage in eukaryotic cells

[0540] This embodiment was performed to verify the RNA-guided cleavage of the TnpB peptide in eukaryotic cells.

[0541] Fluorescent reporter systems are used to identify RNA-guided cleavage. For example... Figure 6 As shown, the system comprises a target sequence (SEQ ID NO: 240) located between the mRFP coding sequence and the GFP coding sequence (SEQ ID NO: 140 and 141), and a TAM. The mRFP and GFP coding sequences are connected out of frame, therefore, GFP will not be expressed if no cleavage occurs in the target sequence. Once cleavage occurs, mRFP and GFP can be connected in frame by repair.

[0542] Construct plasmid groups, each containing a plasmid encoding TnpB (TnpB plasmid), a plasmid encoding the corresponding gRNA (gRNA plasmid), and a reporter plasmid containing a fluorescent reporter system.

[0543] In short, to construct the reporter plasmid, oligonucleotides containing the target sequence and associated TAM were ordered from Tsingke (Beijing, China). The oligonucleotides were then annealed and ligated into a pRGS vector digested with EcoRI and BamHI (see Kim et al., Surrogate Reporters for Enrichment of Cells with Nuclease-induced Mutations, Nature Methods, 2011, 8(11):941-944). The gRNA plasmid was constructed by inserting an oligonucleotide encoding gRNA flanked by EcoRI and BamHI restriction sites (target sequence + backbone as shown in Table 1) into the pUC19 plasmid controlled by the U6 promoter. The TnpB plasmid was constructed by inserting the coding sequence of the TnpB polypeptide (see Table 1) into pcDNA3.1. Maps of the three plasmids are shown below. Figure 7 As shown.

[0544] According to the manufacturer's instructions, use A set of plasmids (120 ng TnpB plasmid + 80 ng gRNA plasmid + 200 ng reporter plasmid) was co-transfected into HEK293T cells (ATCC, CRL3216) using the Invitrogen 2000 reagent. The resulting cells were analyzed by flow cytometry (LSRFortessa, BD Bioscience).

[0545] like Figure 8 As shown, five of the 29 TnpB peptides (ISTfu1, ISDge10, ISAba30, ISAam1, and ISYmu1) exhibit RNA-guided cleavage in eukaryotic cells, meaning they show GFP signals in the presence of both TnpB peptides and gRNA.

[0546] Example 4. TAM preference of ISTfu1 TnpB peptide

[0547] The predicted TAM sequence of the ISTfu1 TnpB peptide is 5'-TGAT, which is similar to the TAM sequence (5'-TTGAT) of ISDra2 TnpB. To further identify the differences between them, a series of reporter plasmids containing the TAM sequence 5'-nTGAT (n = T, A, G, or T) were constructed as described in Example 3.

[0548] As described in Example 3, the test plasmids encoding ISTfu1 TnpB peptide or ISDra2 TnpB peptide and their respective guide RNAs were co-transfected with reporter plasmids into 293T cells, and the resulting cells were detected by flow cytometry.

[0549] The results are shown as a percentage of GFP and normalized to the percentage of GFP in the ISDra2 TnpB / TTGAT group.

[0550] like Figure 9 As shown, the ISTfu1 TnpB peptide can recognize all four TAMs and exhibits higher cleavage efficiency for TAMs TTGAT and CTGAT, while the ISDra2 TnpB peptide can only recognize TTGAT. Furthermore, the ISTfu1 TnpB peptide demonstrates twice the cleavage efficiency of the ISDra2 TnpB peptide.

[0551] Example 5. RNA-guided cleavage of endogenous genes in human cells

[0552] This embodiment was performed to verify the RNA-guided cleavage of endogenous genes in human cells by the TnpB peptide of the present invention. The gRNA backbone used in this embodiment is those listed in Table 1.

[0553] 5.1. Editing ISTfu1 TnpB in hDNMT1

[0554] As described in Example 3, a plasmid encoding the ISTfu1 TnpB polypeptide (SEQ ID NO:7) and a gRNA containing the targeting region (target sequence 1 in hDNMT1) of SEQ ID NO:142 and the backbone of SEQ ID NO:117 was constructed and transfected into HEK293T cells. After incubation at 37°C for 2 days, the transfected cells were collected, and genomic DNA was isolated using an isolation kit (DP201, Bioteke Corporation, Beijing) according to the manufacturer's instructions. Genomic DNA was analyzed by Surveyor assay to identify cleavage efficiency, and genomic DNA from untreated 293T cells was used as a control.

[0555] The surveyor assay was performed according to the 2010 article by Guschin et al. (Guschin et al., ARapid and General Assay for Monitoring Endogenous Gene Modification, Methods in Molecular Biology, 2010, 649:247-256). In short, 100 ng of genomic DNA was used in a 25 μL PCR reaction system, using AccuPrime Taq polymerase (Invitrogen, USA) and primers of SEQ ID NO: 218 and 219. The PCR conditions were as follows: 94℃ for 2 min; 30X (94℃ for 20 s, 60℃ for 20 s, 68℃ for 40 s); 68℃ for 3 min; maintained at 4℃.

[0556] The 6.5 μL PCR product was then denatured, annealed with 3 μL 1X AccuPrime Buffer II, and digested with 0.5 mL Surveyor nuclease (Integrated DNA Technologies, IDT, USA). Samples were run on a 10% acrylamide TBE gel, stained with ethidium bromide for 10 min, rinsed with water, and then exposed to a Bio-rad gel imaging system. Band intensity was quantified using ImageJ software, and genome editing efficiency was calculated using the following formula:

[0557] %genome editing = 100 * (1 - (1 - fraction cleaved)) 1 / 2 ).

[0558] like Figure 10 As shown in Figure A, lanes #1, #2, and #3 show Surveyor nuclease cleavage (indels% of 14.5%, 16.6%, or 20.0%), indicating that the ISTfu1 TnpB peptide can achieve RNA-guided human DNMT1 cleavage in human cells.

[0559] 5.2. Editing ISDge10 TnpB in hTET1, hTET2, and hHPRT

[0560] As described in Example 3, a plasmid encoding the ISDge10 TnpB polypeptide (SEQ ID NO: 17), a gRNA containing the target region of SEQ ID NO: 143 (target sequence 1 in hTET1), 144 (target sequence 1 in hTET2), or 145 (target sequence in hHPRT), and the backbone of SEQ ID NO: 127 was constructed and transfected into 293T cells. A plasmid encoding spCas9 and a gRNA containing the same target region were used as a reference.

[0561] As described in Example 5.1, transfected cells were tested using the primers listed below via a genomic DNA surveyor assay.

[0562] SEQ ID NO:143 SEQ ID NO:220 SEQ ID NO:221 SEQ ID NO:144 SEQ ID NO:222 SEQ ID NO:223 SEQ ID NO:145 SEQ ID NO:224 SEQ ID NO:225

[0563] like Figure 10 As shown in B, the ISDge10 TnpB peptide achieves RNA-guided cleavage of hTET1, hTET, and hHPRT, thereby introducing indels, indicating that the ISDge10 TnpB peptide can achieve RNA-guided cleavage of hTET1, hTET2, and hHPRT in human cells.

[0564] 5.3. Editing ISAba30 TnpB in hDNMT1

[0565] As described in Example 3, a plasmid encoding the ISAba30 TnpB polypeptide (SEQ ID NO:22) and gRNA containing the targeting region (target sequence 2 in hDNMT1) of SEQ ID NO:146 and the backbone of SEQ ID NO:132 was constructed and transfected into 293T cells.

[0566] As described in Example 5.1, the transfected cells were tested by a genomic DNA surveyor assay using primers of SEQ ID NO:226 and 227.

[0567] like Figure 10 As shown in C, the ISAba30 TnpB peptide achieved RNA-guided cleavage of hDNMT1, thereby introducing indels (the percentages of indels for #1 and #2 were 24.9% and 26.0%, respectively), indicating that the ISAba30 TnpB peptide can achieve RNA-guided cleavage of hDNMT1 in human cells.

[0568] 5.4. Editing ISAam1 TnpB in hTET1 and hTET2

[0569] As described in Example 3, a plasmid encoding the ISAam1 TnpB polypeptide (SEQ ID NO:23) and a gRNA containing the targeting region of SEQ ID NO:147 (target sequence 2 in hTET1) or 148 (target sequence 2 in hTET2) and the backbone of SEQ ID NO:133 was constructed and transfected into 293T cells. A plasmid encoding spCas9 and a gRNA containing the same targeting region were used as a reference.

[0570] As described in Example 5.1, transfected cells were tested using the primers listed below via a genomic DNA surveyor assay.

[0571] SEQ ID NO:147 SEQ ID NO:228 SEQ ID NO:229 SEQ ID NO:148 SEQ ID NO:230 SEQ ID NO:231

[0572] like Figure 10 As shown in D, the ISAam1 TnpB peptide achieves RNA-guided cleavage of hTET1 and hTET2, thereby introducing indels, indicating that the ISAam1 TnpB peptide can achieve RNA-guided cleavage of hTET1 and hTET2 in human cells, with indels percentages comparable to or even higher than those of spCas9.

[0573] 5.5. Editing ISYmu1 TnpB in hDNMT1, hDNMT3b, hTET, and hPGK1

[0574] As described in Example 3, a plasmid encoding the ISYmu1 TnpB polypeptide (SEQ ID NO:24) and a gRNA containing the backbone of SEQ ID NO:142, 149 (target sequence in hDNMT3b), 150 (target sequence 3 in hDNMT1), 151 (target sequence 3 in hTET2) or 152 (target sequence in hPGK1) and SEQ ID NO:207 or 210 was constructed and transfected into 293T cells.

[0575] As described in Example 5.1, transfected cells were tested using the primers listed below via a genomic DNA surveyor assay.

[0576] SEQ ID NO:142 SEQ ID NO:218 SEQ ID NO:219 SEQ ID NO:149 SEQ ID NO:232 SEQ ID NO:233 SEQ ID NO:150 SEQ ID NO:234 SEQ ID NO:235 SEQ ID NO:151 SEQ ID NO:236 SEQ ID NO:237 SEQ ID NO:152 SEQ ID NO:238 SEQ ID NO:239

[0577] like Figure 10 As shown in E, the ISYmu1 TnpB peptide achieves RNA-guided cleavage of hDNMT1, hDNMT3b, hTET, and hPGK1, thereby introducing indels, indicating that the ISAba30 TnpB peptide can achieve RNA-guided cleavage of hDNMT1, hDNMT3b, hTET, and hPGK1 in human cells.

[0578] 5.6. Comparison of ISAam1 and ISYmu1 with ISDra2 in human gene editing

[0579] Plasmids encoding ISAam1 TnpB peptide (SEQ ID NO:23) / ISYmu1 TnpB peptide (SEQ ID NO:24) / ISDra2TnpB peptide and gRNAs containing the target regions of the AGBL1-1, APOB-3, EMX1, MECP2, PGK1 and TET1 genes (see Table 2) and the backbone of SEQ ID NO:133 (for ISAam1), SEQ ID NO:210 (for ISYmu1) or SEQ ID NO:158 (for ISDra2).

[0580] Table 2

[0581]

[0582] As described in Example 5.1, transfected cells were tested using the primers listed below via a genomic DNA surveyor assay.

[0583] like Figure 13 As shown, the ISAam1 and ISYmu1 TnpB peptides achieved higher editing efficiency than ISDra2 in most of the tested genes in human cells.

[0584] Example 6. gRNA backbone analysis of TnpB peptide

[0585] The gRNA backbone of the ISDra2, ISTfu1, ISDge10, ISAba30, ISAam1, and ISYmu1 TnpB peptides is designed with "N" nucleotides (referred to as "gN") at the right end of IS, and their sequences are shown in Table 3-8. That is, "N" represents the left end of the gRNA backbone.

[0586] Table 3. gRNA backbone of ISDra2

[0587] g97 153 g115 154 g129 155 g149 156 g174 157 g199 158 g231 159 g248 160 g270 161 g301 162

[0588] Table 4. ISTfu1 gRNA backbone

[0589] g100 163 g118 164 g139 165 g163 166 g183 167 g200 168 g219 169 g240 170 g260 171 g280 172 g298 173

[0590] Table 5. gRNA backbone of ISDge10

[0591]

[0592]

[0593] Table 6. ISAba30 gRNA backbone

[0594] g101 185 g121 186 g140 187 g161 188 g183 189 g204 190 g224 191 g242 192 g261 193 g278 194 g300 195

[0595] Table 7. ISAam1 gRNA backbone

[0596] g100 196 g122 197 g142 198 g164 199 g182 200 g200 201 g218 202 g242 203 g259 204 g280 205 g300 206

[0597] Table 8. gRNA backbone of ISYmu1

[0598]

[0599]

[0600] RNA-guided cleavage of ISDra2, ISTfu1, ISDge10, ISAba30, ISAam1, and ISYmu1 TnpB peptides was performed as described in Example 3, and the results are as follows: Figure 11 As shown.

[0601] Specifically, ISDra2 TnpB can cleave target sequences with a 129nt gRNA backbone ( Figure 11 A); The ISTfu1 TnpB polypeptide can cleave the target sequence with a 139nt gRNA backbone. Figure 11 B); The ISDge10 TnpB peptide can cleave the target sequence with a 122nt gRNA backbone (B); Figure 11 C); The ISAba30 TnpB polypeptide can cleave the target sequence with a 101nt gRNA backbone (C); Figure 11 D); The ISAam1 TnpB peptide can cleave the target sequence with a 100nt gRNA backbone (D); Figure 11 E); ISYmu1 TnpB peptide can cleave target sequences with a 120nt gRNA backbone (E); Figure 11 F). TnpB peptides can also cleave target sequences with a longer gRNA backbone (F). Figure 11 A-11F indicates that a longer gRNA backbone can be used to form RNPs.

[0602] The ISDra2, ISTfu1, and ISDge10 TnpB peptides were analyzed based on their backbones (based on the backbones of SEQ ID NO: 157, 166, and 178, respectively, see...). Figure 10 The right end of the skeleton design of A-10C (right figure) has been modified (see...) Figure 10 A-10C (right figure), and RNA-guided cleavage was performed as described in Example 3. Figure 10As shown in A-10C, substitution of right-terminal nucleotides inhibits RNA-directed cleavage. Substitution of two right-terminal nucleotides greatly inhibits RNA-directed cleavage, while the addition or deletion of one or more right-terminal nucleotides greatly inhibits or even eliminates RNA-directed cleavage, indicating that the right end of the gRNA backbone is crucial for RNA-directed cleavage of the TnpB peptide.

[0603] Example 7. Comparison of editing efficiency between TnpB peptide and Cas nuclease

[0604] This embodiment was conducted to demonstrate that the editing efficiency of the TnpB peptide is superior to that of the small Cas nuclease.

[0605] Specifically, the in vitro and in vivo activities and specificities of the ISAam1 and ISYmu1 TnpB peptides were examined and compared with five well-developed CRISPR-Cas editors, including two optimized Un1Cas12f1 variants (named Un1Cas12f1 and CasMINI), AsCas12f1, an optimized Nme2Cas9 (Nme2-C.NR), and SaCas9. To ensure comparability among these seven systems, despite their differing TAM / PAM requirements, we carefully selected genomic regions where the gRNA for each system could be programmed to target sequences that overlap within a narrow range (see, e.g., Figure 14 ).

[0606] As described in Example 3, plasmid groups were constructed, each group including a plasmid encoding TnpB or Cas editor, a plasmid encoding the corresponding gRNA (gRNA plasmid), and a reporter plasmid containing a fluorescent reporter system. These plasmids were transfected into HEK293T cells and HCT116 cells (ATCC CCL-247), and the genome editing efficiency at 10 genomic sites was tested.

[0607] like Figure 15 As shown in A and 15B, the editing efficiency of ISAam1 and ISYmu1 TnpB peptides is significantly higher than that of well-developed small Cas editors (Un1Cas12f1, CasMINI, AsCas12f1 and Nme2-C.NR), and comparable to that of SaCas9.

[0608] To further reduce the confusion effect of distance between targets, we compared overlapping (>15 bp) gRNAs targeting three endogenous targets in the HEK293T cell line. Previous patterns were largely replicated, with ISAam1 and ISYmu1 TnpB exhibiting higher activity than the Cas12f nuclease along with SaCas9.

[0609] As previously described, rAAV encoding a genome editing system was prepared (see Ran, FA et al. 2015, In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186-191). In short, a nuclease expression cassette driven by the CMV promoter and a gRNA / reRNA expression cassette driven by the human U6 promoter were cloned between ITRs (see...). Figure 16 A) This was used to prepare recombinant AAV2 and AAV8. HEK293T cells were seeded in 150 mm culture dishes 12 hours before transfection; 30 μg of helper plasmid, 15 μg of AAV2 plasmid, and 15 μg of expression plasmid were transfected with polyethyleneimine. The AAV vector was purified three days later. AAV8 for mouse injection was produced by PackGene Biotech Co. at a concentration of 10... 13 gc / ml.

[0610] C2C12 cells (ATCC CRL-1772) were cultured at 5 x 10⁻⁶ cells / year. 4 GC / well seeding was performed on a 48-well plate. AAV2 was seeded at 10... 4 The infection multiple of gc / well was added to the cells. Cells were collected 4 days after transduction for genomic DNA extraction and editing efficiency analysis via next-generation sequencing. The ISAam1 TnpB and Cas9 systems showed significant activity targeting the Rosa26 locus. Figure 16 A). Given that AsCas12f has the lowest average editing activity among Cas12fs, and that Nme2-C.NR generally has lower activity than saCas9, we removed it from in vivo experiments to avoid unnecessarily sacrificing more mice.

[0611] We then individually delivered single AAV8 vectors encoding each of the five different editing systems into mice and analyzed their editing activity in the target organ, the liver. All animal-related experiments described in this study were performed strictly in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Welfare and Research Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences. The C57BL / 6J mouse strain was obtained from Vitalriver. Six-week-old female C57BL / 6J mice were injected via tail vein with 100 μl of 5 x 10⁻⁶ AAV8 vectors. 11 GC. Mice were sacrificed 14 days later, and liver tissue was collected for genome extraction. These in vivo results largely reproduced the aforementioned in vitro data, with the TnpB and SaCas9 systems exhibiting relatively high activity. Figure 16 B).

[0612] To quantify editing specificity or off-target levels, we employed a candidate-based assay in mouse N2a cells and an unbiased whole-genome assay in human HEK293T cells using iGUIDE-seq (see Nobles, CLetal., 2019, iGUIDE: an improved pipeline for analyzing CRISPR cleavage specificity. Genome Biol. 20, 14). In short, 0.5 × 10⁶ cells were transfected using the Lonza 4D system (program CM-130) with 1 μg nuclease plasmid, 500 ng g RNA plasmid, and 50 pmol double-stranded oligodeoxynucleotides (dsODN). 6 HEK293T cells were used. Cells were collected 3 days after nuclear transfection for genome extraction. A genomic library was prepared and sequenced. Specifically, for the Rosa26 and Angptl3 loci in mice, we predicted potential off-target sites for 7 systems, quantified the indel frequencies of the top two off-target sites, and calculated the ratio between off-target and on-target editing. For the human MAPK8 locus, iGUIDE-seq was performed to characterize specificity. Among these three loci, ISAam1 had the lowest off-target ratio for two loci (…). Figure 17 A and 17B), while ISYmu1 also showed a low degree of off-target editing (no off-target editing at one locus, and a second or third low level of off-target editing at the other two loci).

[0613] In summary, in-depth characterization revealed that ISAam1 and ISYmu1 TnpB outperformed the Cas12 variant in both in vitro and in vivo efficiency, while exhibiting performance comparable to the Cas9 variant. Furthermore, their editing specificity was comparable to these Cas12 or Cas9 variants.

[0614] sequence

[0615] SEQ ID NO:1ISEfa4 TnpB amino acid sequence

[0616] MEQLKAYKFRIYPTEEQEIFFAKSFGCVRKVYNLMLDDRKKAYEEVKNDSSKKMTFPTPAKYKKEFPFLKEIDSLALANAQLNLDKAYKNFFRDKSVGFPRFKSKKNPVQSYTTNNQNGTVALIDSKFIKVPKLKSLVRIKLHRQPKGIIKSATISRHSSGKYYISLLCKEEIIELPKTNSTIGIDLGIMDFAILSDGQKIDNNKFTSKMEKKLKREQRKLSRRALLAKNKGTNLFEAKNYQKQKRKVARLHEKVMNQRTDFLNKLSTEMIKNHDIICIEDLNTKGMLRNHKLAKSISDVSWSKFVTKLQYKADWYGRKIIKVDKWFPSSQICSECGHKDGKKSLEIREWTCPVCHTHHDRDINASINILTEGLRLHSMGLA

[0617] SEQ ID NO:2 ISAs26 TnpB amino acid sequence

[0618] MQILKAYKFRLEPTEQQAQRLRQLGGCARYVWNHGLAETKRILDAGEKLPSAFELNRMLTSWKKDPEMAFLAEAYTDNLQQKLKDLHGAWMRCFDKKLAAKAPVFKKKSDNRDAIRFVNFDKYCRLEKGRVKLPAGLAWVKFRQSQPVEGKIKNATVSQAAGHWYISFQVEQETLEPIHPSGSMVGLDAGIAKLATLSDGTVFEPVNSFKTNQTKLARLQRQLSKKVKFSSNWKKQKAKIQRLHSHIANTRRDYLHKVTTTISKNHAMIVIEDLKVASMSKSAAGTTSQPGRNVRAKSGLNRSILDQGWFELRRQLEYKQAWRGGQVLAINPAYTSQQCACCGHTAKDNRQTQSQFKCAACGYEANADVNGARNILAAGHAVLACGGVVQSGRPMKQEPGTARAAITQ

[0619] SEQ ID NO:3 ISCpe2 TnpB amino acid sequence

[0620] MIRKKAYKFRIYPTKKQAELINKTIGCCRFVFNYSLGVQKTKDNYWNIVEEMVQQGYFQENNWKGEFFNKANSIKDIAKLKKSYDWLKEVDSIALQASVENLDAGYDKYYKKTGSRPKFKSKKNEIQSYTTKLVKAKGNVNIEIVGKGIKLPKLGLVKIENSRNVDGNIKRVTVSRTQSGKYFASILCDVNIQELPKIDKKVGVDVGLKTFAVCSDGYEEANPKHFRKAEKRLIKLQRDLARKEYNSKNYHKNRIMIAKLHERIANQRMDFLQKFSTKLIRENQSIAIEDLRVSNMLKNHKLAKVISEASWSEFRRMLEYKAEWYGREIVIAPPDYASSQLCSECGYKNIEVKNLGLREWVCPECGTHHQRDLNASINLEKLIA

[0621] SEQ ID NO:4 ISMma22 TnpB amino acid sequence

[0622] MLKVLRYRVYPTKAQKTTMNNTLELCRWTYNETLALRKDAWESEQKNIGYYDSKKMIPEWKKEKPELKTVHSQVLQEVIKRVDLAFQAFFRRVKTGEKAGYPRFRGYGRYDSFTYTQSGFSLDSDRLTLSKIGDIKIKLHRPVEGEIKRLNVRKMPTGKWFVSFLVETDTPLELQKTGLSVGVDVGIKSFLTLSDGNYVPNPRFFVTEEKFLAKVQRKLSKLEKGTPERAKALKAVQRVHERISNKREDFVQKVSLNLVRNYDLITFEDLNIKGMIKNHKLAKHIADVAWNKLVTITSYKAEWAGKRVELVNPCNTSQMCSGCGEIVKKELSERIHSCPYCGLTLDRDHNAAINIMRLGLQSLQNSGRCPSLQ

[0623] SEQ ID NO:5 ISBce3 TnpB amino acid sequence

[0624] MSQTITVKVKLLPTKEQIQLLEQSSYEYIKVINTLVSEMAEAKKSTKKSTKDVEANLPSAVKNQAIKDAKSVFSTKVKKSKYKVIPILKRPVCVWNNQNYSFDFTHISVPLMVNGKSTRLKVRALLTDKYNRNFNLLTHKLGTLRITKKSNKWIAQISVTIPTNEKTGTKILGVDLGLKVPAVAITDDEKVRFFGNGRQNKYMKRKFRSVRKKLGENKKLNAIRQLDDKEQRWMQDQDHKVSREIIDFATDNNISVIRLEQLTNIRQTARTSRKNEKNLHTWSFYRLAQFIEYKANLVGIKVEYVNPAYTSQTCPKCSEKNKAPDRKYKCRCGFEKHRDIVGAMNIRYATVIDGNSQSA

[0625] SEQ ID NO:6ISAeme8 TnpB amino acid sequence

[0626] MTLRRATKVRIYPTDEQAAFLNAQFGAVRFAYNKALHIQRHMFKRHGISLKPKRDLKPMLAVAKKSRKYGWLKEYDSLALQQAVINLDKAFANFFNPKLKARMPTFKSKRGRQSSYHPNGKVLTAAIQLPKMTPIRAVIHRDIIGVVSSITVSRGPTGKYYASILCEDGCEAPAKPTFITDVTGCDMGLSHYLIESSGKKIANPRHLINASRNLRRKQKALSRKKKGSANRGKARRQLAALHERVANARADFQHKLSHTMVDENQAVIVETLKSANMMKNHKLARAIGDAGWHGFIMKLEYKAQAAGRHLIKLDQWFASSKLCSDCGHKMPDMPLHQRQWVCPACGAGHDRDINAAMNIQQQGILELKAAGHVVSTHGGQR

[0627] SEQ ID NO:7ISTfu1 TnpB amino acid sequence

[0628] MLTGRRYLLAFTPEQEEFAEKIGDACRVVWNTALEQRRQYRRRGAFIGYVEQARQMAEAKKDFPWLAEAPSHTLQQTLRDLERACKTHGTFKVRWRSKRKNAPTFRFPDPKQITVERISRRWGRVKLPKLGWTRFRWTRPLGGQLRNATVLKDGGRWYISFCVEDGLMESAPNGKPPVGVDRGVTVAVATSSGWMRDREFVTPGEAVRLKRLQQQLARQRKGSNRRAATRAKLTKLNARIRARRTDFLAWTANRLTCDHGLVVVEDLNIRNMTASAKGTLAEPGRNVAAKAGLNRAILAKGWGGLLTALEHKARYHGSRILRVPPAFTSQTCHACGHCAPDNRESQAEFRCRACGHQANADVNAAKNILAAGLAVTGRGDLAVGRSVKRQPPKVEAA

[0629] SEQ ID NO:8 ISCco1 TnpB amino acid sequence

[0630] MQITKGFKYRLYPNKEQSLKLSHQCFIYNQAYNICLNLQQEQWLKNKELDKKERTYLSATQIDNQVKLALKARNLEFKSVVAQQARINANRALEQAIKDKNKGFPKFKNSKLAKQSFNWNNQGYQILEFNDKFKIFRLMSMPIKMRYHRALPNNYKLNQITISKTHNKYFVSFSITYDEPSVVLMSSNKIDINRAVGIDMNINEIALSNGALLLTCSKSINKAKYSTTFKRLMRKQSKRVLKAKKDKIKLGSNFRKTQIRLNKIYERASNIKKDLAHKISNQIVKNFDLIVVENLKVKNMTKRAKLKNVKAKSGLNKSILDTSFYQFLSFVEYKAMHNGKVFTKIEPQYTSKACCKCGTIKENLTLKDRVFTCDNCGYVEHRDINASKNILDRGLKSFGLGTSLLDYKQEALRVS

[0631] SEQ ID NO:9 ISSoc3 TnpB amino acid sequence

[0632] MIITHEYRILPSDDQAALMTEWLELLRRQWNDALGQRLDWLTATRCPIDRCSLVSCPLPVSEPPLEPNYYRQAGSLKQIKQLFPAYRGIYAEVLQQNLMRLDKAWKAWREPDSTGKRRGRPRFKKAGELRSFTFPRINCPKAGAHLEGETLRLSKIGSMPVVLHRPLPEGFVPKTCTVVRKADGWYVCITLEDKSVPSPEPVPIKKAVGIDVGLDRFLTTSDGEVVPIPRHYRRAQKHLARQQRQLSRKVKGSANWKRQATKVACLQLHVARQRKAFHYQVAHWLVEQYDLLVVEDLNVRGLARTRLAKSILDAAWGRFLDILTAVAVKRGKQVLRVDPRGTSQNCCVCEERVPKTLSERVHDCPRCGSWDRDLNAAIEILKRGLRAVGLPLSGCGGSWFTSPLKQQLREVILGSSRLQPVRA

[0633] SEQ ID NO: 10 ISTel2 TnpB amino acid sequence

[0634] MKARYRYRFYPTDQQRQRLAQLFGCVRVVWNDALAICKQSDSLPKTSELQKLVITQGKKTPERQWLSDVSNVPLQQSVADLGVAYKNFFDSIKGRRKGKKINPPRFKKKTERQSARFTTYGFSIKGEEVYLAKIGNVRPIWSRKLPSKPSSVTVIKDAANRYFLSFVVEIDPVHQPASNPSIGIDLGIKAFATLSTGEKIDGPDYSKLDKKIRRKQRKLARQVKGSQRREKTRLQIAKLHSRISDIRRDFLHKVSTRVVIENQVITLEDLNVSGMVKNRKLARAISLQGWREFRVLVEAKCNKLNREFIVIDRWEPTSQTCSKCGFKWGKVDLSVRSVVCLNCGTEHDRDENAARNIEKVGIGHCHDYKWTQRGSKTTSVASPSEASRIIAL

[0635] SEQ ID NO: 11 ISNsp3 TnpB amino acid sequence

[0636] MKTLKFKLYQHKRNRFLKRMINASGVIYNHCIALHKRYYRMWGKHLSCAKLQAHIAKLRKRNPFWQTVGSQAVQDICQRIEKAYQLFFKHNKKGVRPPGFKKVKKYKSFTLKQAGYKFLGGNRVKIGNRVYQFWKSRQIEGTVKTLTIKRTPLGELFVVVVVDDGIKPEIEIKTGKIAGFDFGLKTFLTCSDGTKIDSPQFFKQSLNAITKASRQHSKKLKGSANRKRARLNLVRKYEDISNRRRDWFWKLAYELTDKFDVLCFETLNLKGMQRRWGRKISDLAFGEFLEILEWVAKKKNKLIVFIDQWYPSTKTCSHCGHVLESIDLSVRSWSCPSCQSVNERDENASKVICAVGASTVGLGDVRLATPAIAV

[0637] SEQ ID NO:12 ISCbt1 TnpB amino acid sequence

[0638] MAKSKTSSYILTLKLKTEKYQEDILNKRLEISRNIYNSCLGEILKRYKHTRELKDYRKEYKNIIKMSKDKERSKKFNELNKKYDLTEYSLHTYVKPIQKHFKDNVDSFTAQKIATRVFNSFQKLMFHQAKRVYFKRYGELNSVEGKSNKTGIRFQDNSLIWNGLKIPVIIKTNDIYVQMSLENRVKYCRIIRRIVKGKIKFYVQLILEGIPPMKINKTTGEIKNKIGKGNVGIDIGTRTIAVSSENDVKLLELAPEIDNIENQKRILNRKLDRQRRANNPNKYNEDGTINRNNKDKWIKSNRYIKTQNKLKEIQRKQAEIRKQSHYRLINRLLLLGNRFLVETMNYKGLQARVKETTINEKTGRFNKKKRFGKSLANKAPSMFLTMLDNKLKWNNTQLFKIDTKKCKASQYNHFTNEYCKKELKDRWNEDIQIQRDMYSAFLIMNVVGKTLDKIDRDLCIETYDNFRRLHDKEIERLKELKKNGYKLISSMGI

[0639] SEQ ID NO:13 ISMac7 TnpB amino acid sequence

[0640] MMKAFKFRLYPTTTQADQLNQHIGSCRFVYNWALDQKIKTYEQTGKSISRFDLNKLIPALKTSNEWLGEVNSQSLQGMTKQVESAFTRFFREKNGFPKFKSKKNPIQSFPVPQHYSVNFENHTVKLPKIGEIKAVLHRKFDGELKTARVSRTCKGHYYISVLVEDGKELPEKQGFSESTTVGIDVGIKDFAVLSTGEKIENPKYLKNSLQRLKVLQKRVSRKQKGSKNRAKARQRLAVLHDKITNQRNDFQNKLSFRLVSENQAVALETLNVKGMVKNHHLAQAISDSAWSSFVTKLEYKAEWFGKGVLRIGQFEPSSKLCNVCGYHNKELQLKDREWTCPDCKTKHDRDINAAINIKKFALIDQNLIGL

[0641] SEQ ID NO:14 ISEc46 TnpB amino acid sequence

[0642] MLRATKVRIYPTPEQAEFINAQFGAVRFAYNKALHIKKHSYQRHRVNLNPRKDLKPLLAVAKKSRRYGWLKEYDSIALQQAVINLDVAFSNFFNPKLKAGKPTFKSKHGRQSSYHCVGIKVLDGAIKIPKILPIEARLHREITGMLKSITLSRSATGKYYASLLCDDGMEAPAKPALISNVTGLDMGLAHFVIKSDGNKIASPRHLINASRNLRRKQKALSRKKKGNANRRKARIQLAAVHERVASARADFQHKLSRAIVDENQAVIVETLKTANMMKNHHLARAIGDAGWSGFITKLEYKATEKGVHLVKLDQWFASSKTCHCCGHKMSEMPLHKRIWQCPECGVEHDRDINAALNIKRKGILELQAAGLVVSAHGGQHKSVTQTVAA

[0643] SEQ ID NO:15 ISSen6 TnpB amino acid sequence

[0644] MMLILKAYKFRLEPTPEQSQRLRQLCGCARFVWNLGLAETKRILGSGEKLPSAFELNRMLTVWKKMPGHIFLQDAYTDNLQQKLKDLHTAWKRCFDKKLAAKAPVWKRKNEGRDSIRFVNFEKYCRLENRRVKLPSGLGWVKFRQSQRVNGKIKNATISQLAGQWYISFQVEVETAEPNHTSTTIVGLDAGVTKLATLSDGTVYQPVNSFKASQRKLATLQRQLSRKVRFSSNWQKQKRKVQHLHSHIANIRRDYLHKVTSEISKNHAMIVIEDLKVSNMSKSAKGTAEQHGRNVKAKSGLNRSILDQGWYEMRRQLEYKQLWRGGQVLAVPPAYTSQRCACCGHTAKENRLSQSKFVCQACGYTANADVNGARNILAAGHAVLACGGMVQSGRPSETGTRR

[0645] SEQ ID NO:16 ISHahl1 TnpB amino acid sequence

[0646] MIQRKATYRLYPTAEQLAALEHQLWVHCLLWNEALAERRWAWSERQESLGFSAQCKRLTEWRSQSKLLSGLNAQSEQVTLKRLDLAFQHFFRRVKQGETPGFPRFKSLHRFKGWGYKTHGDGWRLLTNEQMQHGTLRLSGVGAVAVRGRPRTPGTPKTCEIIKRGDRWYASITLNCEPVRECGEKMGGLDWGLETFATVATAQGAETIDNPRHLAGTLDAIRGVQREISRKEEAAKKASGRSRGFPISNRLRKAYDELRRLHRKVANQRHDFLHQVSARLIKEFSALGLEALSIRNMTAKGGQHKRGLNRGILDAAGGAFHQLLGYKAEEAGAWAVEAPTRQIKPSQTCHACGQQEKKPLGQRWHSCPCGASCSRDENAARVLLAWLERSLSGREPADAWREVRPGHPLDESALPSKRETHAIA

[0647] SEQ ID NO:17 ISKpn69 TnpB amino acid sequence

[0648] MLIQKAYKYRLNPTDAQAQRLRQMCGCARFVWNYALAETQRILEAGQKLPSAFELNKMLTGWKKLPDLAFLSDAYTDNLQQKLKDLHGAWIRCFDKKLAAKAPVFKRKTHGNDSVRFVNFDKYCQLEYRRVKLPSGLGWVTFRQSRAVVGKIKNATVSQHAGGWYISFQVEMEVEQPAHPSTSAVGLDAGICKLVTLSDGTVFEPVNSFKQNQSRLARLQRQLARKVKFSANWKKQKARISRLHSHIANIRRDYLHKVTTTISKNHAMIVIEDLKVSNMSKSAAGTVDEPGRNVAAKSGLNRAILDQGWYEMRRQLEYKQRWRGGEVLAVNPAYTSQKCARCGHTVKENRQSQAVFVCVACGYEANADINGAQNILAAGHAVLSGINPGRARKAA

[0649] SEQ ID NO: 18 ISDge10 TnpB amino acid sequence

[0650] MTTHRKVYRYRIEPTPVQESKLYMLAGSRRFVFNWALARRREHYAETGKTLGYNAQAGELTALKNQEETSWLKESDSQLLQQALKDVERAFVNFFEKRARFPRFKSKKTDTPRFRIPQRVRIEGSRVYVPKVGWVKLRKSQEIEGKTKSATFKREADGHWYVLLVSEFEMPDVPLPPVPESEVVGIDLGLKDFYVLSDGGRKEAPRFARKGQRKLRRAARRHSKCTRGSNRKAKAKRKLARVHRQIANQRKDFVHKATSGLVQQYQGFCIENLSIKGMAKTKLSKSVLDAALGEFRRQLAYKAQWHRKWLAVIDRWFPSSKLCGECGSINADLTLSDREWTCECGAVHDRDLNAARNIKREGLSQIVVAGHAETLNARGEGVRPAIAGSPR

[0651] SEQ ID NO: 19 ISKpn85 TnpB amino acid sequence

[0652] MLIQKAYKFRLNPTDVQAQRLRQLCGCARFVWNYGLAETQRILETGQKLPSAFELNKMLTGWKKLPDLAFLSDAYTDNLQQKLKDLHGAWKRCFDKKLAAKAPVFKRKSHGNDSVRFVNFDKYCQLEYRRVKLPSGLGWVTFRQSRDIVGKIKNATVSQHAGSWYISFQVEMEVDQPAHPSTSAVGLDAGVSKLATLSDGTVFEPVNSFKQNQSRLARLQRQLARKVKFSANWKKQKARISRLHSHIANIRLDYLHKVTTTVSKNHAMIVIEDLKVSSMSKSAAGAADMPGRNVAAKSGLNKAILDQGWYELRRQLEYKQSWLGGQVLAVNPAYTSQKCACCGHRSKENRKSQANFVCLECGYKENADINGARNILAAGHAVLACEVNGASMPSAAGTKRLSNQLATQ

[0653] SEQ ID NO:20 ISNsp2 TnpB amino acid sequence

[0654] MKTLKFKLYQHKRNRFLKRMINAGGAIYNHCIALHKRYYRMWGKHLNCAKLQAHIAKLRKRNPFWQTVGSQAVQDICQRIEKAYQLFFKHNKKGVRPPGFKKVKKYKSFTLKQAGYKFLDGNRVKIGSKIYQFWKSREIEGTVKTLTIKRTPLGELFMVVVVDEGNKPETKFMTGKIAGFDFGLKTFLTCSDGTTIESPQFFKQSLNAIKKASRQHSKKLKGSANREQARKNLVRRYEDIFNRRRDWFWKLAHELTDKFDVLCFETLNLKGMQRLWGRKISDLAFGELLQILEWVAKNKNKLVVFIDPWYPSSKTCSSCGHILENLDLSIRRWRCPSCQSENDRDGNASLNIKRVGSSTLGVGDVRQSQTAISV

[0655] SEQ ID NO:21 ISAba30 TnpB amino acid sequence

[0656] MKRLQAFKYEMVPNGQQERQMRRFAGSCRFVYNKALALQKERYERGEKKLGYAGLCKLLTEWRNSSEMAWLADAPVHPLQQTLKDLERAYSNFFAKRADFPRFKKKGCHSSFRYPDPKQIKLEQHNSRIFLPKLGWIRYRKSRDVLGAVKNVTVSLSGGKWFVSIQTEREVEQPIPQGGAVGIDMGIARFATISDGKFYAPLNSFKRHEKRLRCAQQALSRKVKFSNNWQKAKARIQRIHSRIGNARRDFLHKTSTAISQNHAMVCIEDLQVRNMSKSAAGTPEQPGRNVRAKSGLNKSILDQGWFEFRRQLDYKLAWNGGWLVAVPPKNTSRTCPCCAHVSADNRQTQERFECIECGFEGNADVVGAINVLRAGHARLACEVSDAVGSPAAGTHRSDSDQAHCRV

[0657] SEQ ID NO:22 ISRor9 TnpB amino acid sequence

[0658] MHRATKVRIYPTQEQTELLEKQFGAVRFAYNKALYIRSHWYKKHGVSLNNAKHIQPLLPIAKKSRRYSWLKAADSNALQVSLQNLDRAFQNFFEKRAKYPKFKRKHGPQSSYHCSSLKITADTIKIPKISPIRANIHREIVGKVKSITISRAPTGKYYASILADDGQEAPAKPTYITQLTGYDIGLKDYLIRDDGKKTPNPRHLVNAYRRLRRDSKALSRKQKGSANRRKARIRLAAQHDRVANARNDFQHKLSRNIIVENQAVIVETLKTANMMKNKRLARHIGDAAWSGFVAKLAYKAEASGVHLVKLDQWFASSKTCHCCGHKMAEMPLKVREWQCPVCLTHHNRDINAAMNIRLKGITELMAAGHVVKAHGGLRKSVITTVAAYEVGSPAL

[0659] SEQ ID NO:23 ISAam1 amino acid sequence

[0660] MVNKSYKFRLYPTKEQEQLLAKTFGCVRFVYNKMLEERIQIYEKFKDDKEALKKQTFPTPAKYKKEFPWLKEVDSLALANAQLNLQKAFQNFFSGRAGFPKFKNRKAKQSYTTNVVNGNIQLSDGYIKLPKLKWVKFKQHREIPAHHIIKACTIKKTKTGKYYVSILTEYEHQPVPKEIQTVVGLDFSMNGLFVDSEGKRANYPRFYRQALEKLAKEQRILSRRKKGSNRWHKQRLKVAKLHEKIANQRKDFLHKKSYELAKQYDCVVIENLNMKGMSQVLNFGKSVHDNGWGMFTTFLQYKLEEQGKKLIKIDKWFPSSKTCSCCDQVKESLSLSERTFRCECGFESDRDVNAAINIKHEGMKRLAIA

[0661] SEQ ID NO:24 ISYmu1 amino acid sequence

[0662] LLQHKAYEYRIYPDKKQETLIAKTIGSSRYVYNHFLELWNKEYEETGKGLTYYACSKLLTKLKRDPETVWLCEVDKFSLQNSLRNLSDAFSRFFKGQNEHPQFKSKKSPRQSYTTQYTNNNIAVSGNCLKLPKLGLVKFADSREMKGRILNATVRRKSSGKFFVSILCEEEICELPKTDSSVGIDLGIIDFAVMSDGSRHDNNHFTRQMEERLRREQRKLARRALAAEKRGISLSEARNYQKQRRKVARLYEKVANQRKEYLNKLSTEIVKNHDIICIEDLNVKGMMRNHKLAKSISDVSWTSLVSKLQYKASWYGKEVIRISRWFPSSQICSECGHKDRKKPLHVREWTCPVCHAHHDRDVNAARNILAEGLRIRALTPGS

[0663] SEQ ID NO:25 ISCytsp1 amino acid sequence

[0664] MTKQNKAYKFRLYPTDEQAFIIRKTFGCVRFVYNKMLAERIETYESLKEDKEALKKIKHPTPAKYKKEYEWLKEVDSLALANAQLNLDKAYKAFFKGNVKFPTFKRKRHKQSYTTNVVNGNIQLLDGHIKLPKLKLVKIKQHRKIPSEYKIKSCTLSMTASGKYYISILAEYEVEIERKEVKKVVGLDFAMDGLFVDSEGEKANYPKFYRQKLEKLAKEQRKLSRKKKGSSNWNKQRIRVANIQEKVANQRKNFLHHQSKKLVANYDAVVIEDLDMKGMSQALKFGKSVADNGWGMFTSFLQYKLKEQGKQLIKIDKWFPSTQTCSHCGNTQLMPMNIRIYVCSCGLNLDRDYNSALNIKKEGIRLLASA

[0665] SEQ ID NO:26 ISCvi1 amino acid sequence

[0666] MNKGVKFRAYPNKEQQNLINRTLGCCRLIYNKGLAMRNDAYSNGEKIGYNQTSAMLTNLKKQADFAFLKDVDSIALQQSLRDLDRGFKNFFEKRARHPQFKSKHNNHQSYRTINQGDNIRIIGKYIKLPKLGYVKIKQSMEVGHINNVTVERTPTGKYFVVLNVDFEPEFRPNAGCMIGIDVGIKEFYSDSNGNVVNNPKYLEKSMRKLAREQRRLSRKQKGSNNRNRQRVKVAKVHEKITNQRNDFLQKQSTILVSENQTICIEDLNVKGMIRNHKLAQHIASCSWSKFFAMLEYKAIWYGNDIIKVPTMYPSSQTCSCCGYKNPFVKNLSIREWECPNCHAKHDRDTNASITILNKGLSIA

[0667] SEQ ID NO:27 ISCvi2 amino acid sequence

[0668] MNKAIKYRVHPTTEQSVMFAKTFGCCRKVYNLMLSDKIDGYKATGKFPTVTPAKYKNDYPYLREVDSLALANKQMDLQEAFRNTFSKSRKKKNGFPKFKSAKHSRKSYTTNNQHGTVAIIDNKYIKLPKIGKVKAVIHRIPDSDWIVKSATVSQESDGKYYISVLFEFDNAINLYVADKTNAIGLDYTSNGLYVDSNGNVGTNHKYYRESHDKLAKAQRRLSRMQGSKKHEIKSNNYIKQLRKVNKIHRYIANQRLDNLHKISTKIANSYDVVCVESLNMRSMANHGFGNGKATLDNGYGMFLSMLEYKLSDRNKYLIKVDKWFPSSQICHCCGKIHPEMKNLTIRTMKCDCGLTMNRDQNAAINILREGLRLLTKVA

[0669] SEQ ID NO:28 ISAepa1 amino acid sequence

[0670] MAKQNKAFKFRLYPNKEQKIQLAKTFGCVRFVYNKMLEERKETYEKFKNDKETLKQQAFPTPAKYKDEFPFLKEVDSLALANAQINLQNAYKNFFEGRAEFPKFKSKKNKQSYTTNMVNGNIKLENGHIKLPKIKKPMKMNRHREIPADYKIKSCTISKTKTGKYYISILTEYEKDIQSVKIQKVVGLDFAMDGLYVESEQGEKANYPRYYRQALDKLAKAQRILSRRKKGSARWEKQRLKVAKLQEKIANQRKNFLHHKSKELASTYDAVIIEDLDMKGMSQALNFGKSVHDNGWGMFTTFLEYKLKEQGKQLVKIDKWFPSTKKCSCCGKEKEMPLSKRVYECSCGFVLDRDHNSAINIKNEGLRILA

[0671] SEQ ID NO:29 ISBth16 amino acid sequence

[0672] MTKQNKAYKFRLYPTKDQAHLMRKTFGCVRFVYNRMLAERKEAYEKHKDDKDQLKKQKLPTPAKYKAEFEWLKEVDSLALANAQLNLQTAYKNFFRGQNDFPTFKSKKDRKSYTTNVVNGNIILLNGHIKLPKLKMVRIKQHREIPQGHIIKSCTISMTPTGKYYVSILTEYEKEIVQKEVETVVGLDFAMDQLYVSSEDERANYPKFYREMLDRLAKAQRVLSRRTKGSGRWNKQRIRVAKLHEKVANQRKNFLHHKSKELATHFDVVAIEDLNMKGMSQALHFGKSVADNGWGMFTSFLAYKLHEQGKQLVKIDKWFPSTKTCSSCGNMKNMSLSERVYSCICGVNLDRDYNAAINIKNEAIRLLALV

[0673] SEQ ID NO:30 ISEfa4 TnpB nucleotide sequence

[0674]

[0675] SEQ ID NO:31ISAs26 TnpB nucleotide sequence

[0676]

[0677] SEQ ID NO:32ISCpe2 TnpB nucleotide sequence

[0678]

[0679] SEQ ID NO:33ISMma22 TnpB nucleotide sequence

[0680]

[0681] SEQ ID NO:34ISBce3 TnpB nucleotide sequence

[0682]

[0683] SEQ ID NO:35ISAeme8 TnpB nucleotide sequence

[0684]

[0685] SEQ ID NO:36ISTfu1 TnpB nucleotide sequence

[0686]

[0687] SEQ ID NO: SEQ ID NO: 37ISCco1 TnpB nucleotide sequence

[0688]

[0689] SEQ ID NO:38ISSoc3 TnpB nucleotide sequence

[0690]

[0691] SEQ ID NO:39ISTel2 TnpB nucleotide sequence

[0692]

[0693] SEQ ID NO:40ISNsp3 TnpB nucleotide sequence

[0694]

[0695] SEQ ID NO:41ISCbt1 TnpB nucleotide sequence

[0696]

[0697] SEQ ID NO:42ISMac7 TnpB nucleotide sequence

[0698]

[0699] SEQ ID NO:43ISEc46 TnpB nucleotide sequence

[0700]

[0701] SEQ ID NO:44ISSen6 TnpB nucleotide sequence

[0702]

[0703] SEQ ID NO:45ISHahl1 TnpB nucleotide sequence

[0704]

[0705] SEQ ID NO:46ISKpn69 TnpB nucleotide sequence

[0706]

[0707]

[0708] SEQ ID NO:48ISKpn85 TnpB nucleotide sequence

[0709]

[0710] SEQ ID NO:49ISNsp2 TnpB nucleotide sequence

[0711]

[0712] SEQ ID NO:50ISAba30 TnpB nucleotide sequence

[0713]

[0714] SEQ ID NO:51ISRor9 TnpB nucleotide sequence

[0715]

[0716] SEQ ID NO:52ISAam1 TnpB nucleotide sequence

[0717]

[0718] SEQ ID NO:53ISYmu1 TnpB nucleotide sequence

[0719]

[0720] SEQ ID NO:54ISCytsp1 TnpB nucleotide sequence

[0721]

[0722] SEQ ID NO:55ISCvi1 TnpB nucleotide sequence

[0723]

[0724] SEQ ID NO:56ISCvi2 TnpB nucleotide sequence

[0725]

[0726] SEQ ID NO:57ISAepa1 TnpB nucleotide sequence

[0727]

[0728] SEQ ID NO:58ISBth16 TnpB nucleotide sequence

[0729]

[0730] SEQ ID NO:59ISHaal1 TnpB nucleotide sequence

[0731]

[0732] SEQ ID NO:60ISNsp7 TnpB nucleotide sequence

[0733]

[0734] SEQ ID NO:61ISTva1 TnpB nucleotide sequence

[0735]

[0736] SEQ ID NO:62ISClsp11 TnpB nucleotide sequence

[0737]

[0738] SEQ ID NO:63ISVgsp2 TnpB nucleotide sequence

[0739]

[0740] SEQ ID NO:64ISBsp10 TnpB nucleotide sequence

[0741]

[0742] SEQ ID NO:65ISCath1 TnpB nucleotide sequence

[0743]

[0744] SEQ ID NO:66ISPqu1 TnpB nucleotide sequence

[0745]

[0746] SEQ ID NO:67ISViha1 TnpB nucleotide sequence

[0747]

[0748] SEQ ID NO:68ISCno2 TnpB nucleotide sequence

[0749]

[0750] SEQ ID NO:69ISNsp8 TnpB nucleotide sequence

[0751]

[0752] SEQ ID NO:70ISMba21 TnpB nucleotide sequence

[0753]

[0754] SEQ ID NO:71>IS605 TnpB nucleotide sequence

[0755]

[0756] SEQ ID NO:72>ISLj o5 TnpB nucleotide sequence

[0757]

[0758] SEQ ID NO:73>ISHla19 TnpB nucleotide sequence

[0759]

[0760] SEQ ID NO:74>ISSto1 TnpB nucleotide sequence

[0761]

[0762] SEQ ID NO:75>ISH12 TnpB nucleotide sequence

[0763]

[0764] SEQ ID NO:76>ISH35 TnpB nucleotide sequence

[0765]

[0766] SEQ ID NO:77>ISHal1 TnpB nucleotide sequence

[0767]

[0768] SEQ ID NO:78>ISHP608 TnpB nucleotide sequence

[0769]

[0770] SEQ ID NO:79>ISHuh1 TnpB nucleotide sequence

[0771]

[0772] SEQ ID NO:80>ISHma12 TnpB nucleotide sequence

[0773]

[0774] SEQ ID NO:81>ISClte2 TnpB nucleotide sequence

[0775]

[0776] SEQ ID NO:82>ISNamo19 TnpB nucleotide sequence

[0777]

[0778] SEQ ID NO:83>ISH34 TnpB nucleotide sequence

[0779]

[0780] SEQ ID NO:84>IS606 TnpB nucleotide sequence

[0781]

[0782] SEQ ID NO:85>ISMba17 TnpB nucleotide sequence

[0783]

[0784] SEQ ID NO:86>ISCsa5 TnpB nucleotide sequence

[0785]

[0786] SEQ ID NO:87>ISHwa17 TnpB nucleotide sequence

[0787]

[0788] SEQ ID NO:88>IS609 TnpB nucleotide sequence

[0789]

[0790] SEQ ID NO:89>ISMma19 TnpB nucleotide sequence

[0791]

[0792] SEQ ID NO:90>ISH22 TnpB nucleotide sequence

[0793]

[0794] SEQ ID NO:91>ISHli11 TnpB nucleotide sequence

[0795]

[0796] SEQ ID NO:92>ISHma17 TnpB nucleotide sequence

[0797]

[0798] SEQ ID NO:93 ISCysp14 TnpB nucleotide sequence

[0799]

[0800] SEQ ID NO:94 ISSoc6 TnpB nucleotide sequence

[0801]

[0802] SEQ ID NO:95>ISCbt2 TnpB nucleotide sequence

[0803]

[0804] SEQ ID NO:96>ISTac1 TnpB nucleotide sequence

[0805]

[0806] SEQ ID NO:97>ISEc41 TnpB nucleotide sequence

[0807]

[0808] SEQ ID NO:98>ISFac4 TnpB nucleotide sequence

[0809]

[0810] SEQ ID NO:99>ISSoc10 TnpB nucleotide sequence

[0811]

[0812] SEQ ID NO:100>ISHma17 TnpB nucleotide sequence

[0813]

[0814] SEQ ID NO:101>ISDka2 TnpB nucleotide sequence

[0815]

[0816] SEQ ID NO:102>ISHsi2 TnpB nucleotide sequence

[0817]

[0818] SEQ ID NO:103>ISHru2 TnpB nucleotide sequence

[0819]

[0820] SEQ ID NO:104>ISChy9 TnpB nucleotide sequence

[0821]

[0822] SEQ ID NO:105>ISAsp6 TnpB nucleotide sequence

[0823]

[0824] SEQ ID NO:106>ISHsal13 TnpB nucleotide sequence

[0825]

[0826] SEQ ID NO:107>ISCARN6 TnpB nucleotide sequence

[0827]

[0828] SEQ ID NO:108>ISFsp4 TnpB nucleotide sequence

[0829]

[0830] SEQ ID NO:109>ISHlsp1 TnpB nucleotide sequence

[0831]

[0832] SEQ ID NO:110>ISMmu1 TnpB nucleotide sequence

[0833]

[0834] SEQ ID NO:111>ISEfa4 gRNA backbone 213nt

[0835] GTATCAATATTCTGACCGAAGGTCTAAGACTACATTCTATGGGATTGGCTTAGATCCAATCATCAAGAACCGTAGGAACTACGGGGATAGCTTGGTAAATAAGAGACACCGCTGTTAGTAAAGAAATACGCTATCAAGTATGCTCTATTCCCAAGAAGCTCCCACTTCAAGCGTTAAGAACCGTCAGGTTTAGCTAAGTGGTGAGTAGTTCAC

[0836] SEQ ID NO:112>ISAs26 gRNA backbone 218nt

[0837] GCCAGTTTAAATGCGCTGCGTGTGGTTATGAGGCGAACGCCGATGTGAACGGCGCTCGCAATATTTTAGCGGCGGGACACGCCGTTTTAGCCTGTGGAGGCGTGGTGCAATCAGGCCGCCCGATGAAGCAGGAACCCGGTACTGCGCGAGCAGCTATCACCCAGTAATGGGAATCCCCCTCCTGTGACGCGAAGCGCTAAGGTGGGGGAGGATGTCAA

[0838] SEQ ID NO:113>ISCpe2 gRNA backbone 232nt

[0839] GAATGTGGCTATAAGAATATAGAAGTTAAAAATCTAGGTCTAAGAGAATGGGTATGTCCCGAATGTGGAACACACCATCAAAGAGACCTTAATGCTTCAATAAACTTGGAAAAGCTGATAGCTTAAACTATTGGTATCCAACGAGGTCGGAACGCCCTTGTTAGCTTGGTTAATCTTACACCGTTAGGTGTGTTGCCCAAGAATCTTGGCATTTCAATGCCAAGTAGTTCAA

[0840] SEQ ID NO:114>ISMma22 gRNA backbone 195nt

[0841] GTAATACATCACAAATGTGCTCTGGATGCGGTGAAATAGTTAAAAAAGAACTATCAGAGAGAATACACAGTTGTCCTTACTGTGGTCTTACTCTTGATAGAGATCACAATGCCGCTATTAACATTATGAGATTGGGATTACAATCTCTCCAGAATTCTGGTAGATGCCCGTCACTTCAGTGATGGGAGTAGTCAC

[0842] SEQ ID NO:115>ISBce3 gRNA backbone 249nt

[0843] GTGCCGATGTGGATTTGAGAAACATCGTGATATTGTTGGGGCGATGAATATTCGCTACGCAACTGTGATTGATGGTAACAGTCAATCAGCCTAAGATGCTATACGCACTGTCTTAGGAGGGGCAATGAGATGCCCTCATCTTGAAGTCTGTTCAAAACAGAAATGGACTGCGAACGTTTAGTCATTCAAGAATCCCACCCGTCAATCCGTAAGGATTAGGGCTTGCGACTTTAGTCGTGGGAGTCTCAA

[0844] SEQ ID NO:116>ISAeme8 gRNA backbone 224nt

[0845] GCCGGACATGCCGTTGCATCAGCGGCAGTGGGTATGTCCAGCGTGTGGGGCAGGGCATGACCGCGACATTAACGCGGCCATGAATATCCAGCAACAAGGCATATTGGAATTAAAGGCGGCGGGACACGTCGTTTCTACCCATGGAGGCCAGCGTTAATCTGCCAATTCGGCAGTAGCGGCCTAAGAAGTGGGAAGCCTCGCCGCTTGCGGCGGGGAGCAGTCAC

[0846] SEQ ID NO:117>ISTfu1 gRNA backbone 219nt

[0847] GTGAAAGCCAAGCGGAGTTCCGGTGCCGCGCCTGCGGCCACCAGGCCAACGCCGACGTGAACGCCGCGAAGAACATCCTCGCCGCCGGGCTGGCGGTGACAGGGCGTGGAGACCTCGCCGTTGGGCGGTCTGTGAAGCGCCAACCACCCAAGGTAGAGGCAGCGTGAACGCGCCTCAGCCACGGGAATCCCCCGCCTTCAGGCGTGGGGAGGAAGTCAA

[0848] SEQ ID NO:118>ISCco1 gRNA backbone 201nt GATAGAGTTTTTACTTGCGATAATTGTGGATATGTAGAACATAGAGATATAAATGCAAGTAAAAATATCTTGGATAGAGGCTTGAAGTCGTTTGGGCTAGGAACTAGCCTATTAGACTATAAACAAGAAGCCTTGCGTGTTAGCTGACATTGGTTGGCTTTAGTAAGGAAGCACCTACTTCTATAAGTAGGTGTGGTTCAC

[0849] SEQ ID NO:119>ISTel2 gRNA backbone 203nt

[0850] GATCTATCCGTTCGCTCGGTCGTATGTCTAAATTGTGGTACAGAGCACGATAGAGACGAAAATGCTGCAAGAAACATAGAAAAAGTCGGGATAGGGCATTGCCACGACTATAAATGGACGCAGAGGGGGAGTAAGACTACTTCGGTAGCATCACCCAGTGAAGCGTCAAGAATCATCGCACTTTAGGCCGGTGAGTATGTCAA

[0851] SEQ ID NO:120>ISNsp3 gRNA backbone 219nt

[0852] GTTCTCACTGTGGTCATGTCTTAGAGAGTATTGATTTGTCTGTTCGGTCGTGGAGTTGTCCGTCCTGCCAGTCAGTGAACGAGAGAGACGAAAACGCCTCTAAGGTAATTTGTGCAGTCGGGGCATCGACTGTTGGGTTAGGTGATGTCAGACTGGCTACGCCAGCAATCGCTGTTTGAGCCTAGAATCCCCCGAATTATATTCGGGGGAGTATGTCAA

[0853] SEQ ID NO:121>ISCbt1 gRNA backbone 270nt

[0854] GAGATTACACGACAAAGAGATAGAAAGATTAAAAGAATTAAAGAAAAATGGATATAAATTAATATCCAGTATGGGGATATAAAATCTATATAGGTTTAGACATGAGCCTTATACTAATGTTAATCATTTATTAAAATGATAATTAGTGAAAGTCTTAGAGAAACTAACTAGTATTTATATGTTGTAATTTATTTGAAGTTTAAATAAATGAGAGTATAAAAGAAGTTAATGTATCTAAGAACCCCAAGGCTTTAGTCTTGGGAGTGTCAG

[0855] SEQ ID NO:122>ISMac7 gRNA backbone 208nt

[0856] GACAGAGAATGGACTTGTCCTGATTGTAAAACAAAACATGATAGAGACATAAATGCCGCTATCAATATCAAGAAATTCGCTCTCATAGATCAAAATCTAATTGGATTGTAACACCTACGGAACGTGGGGAAGAGCTTGGGGACTTGCCCTCAATAGAGGGAAGAATGAACCAAGAAGCAACTCAGTCTTTAGCTGAGTGGTAGTTCAC

[0857] SEQ ID NO:123>ISEc46 gRNA backbone 220nt

[0858] GAGATGCCATTACATAAGCGCATCTGGCAGTGTCCAGAGTGTGGAGTTGAACATGACCGCGATATCAACGCGGCGCTCAACATCAAACGCAAGGGCATACTGGAATTACAAGCGGCGGGACTCGTCGTTTCTGCCCATGGAGGCCAGCATAAATCCGTCACACAGACGGTTGCGGCTTAAGAAGTGGGAAGCCTCGCCCGACAGGGCGGGGAGCAGTCAC

[0859] SEQ ID NO:124>ISSen6 gRNA backbone 219nt

[0860] GTAAATTTGTGTGTCAGGCATGCGGATATACAGCGAACGCTGATGTAAACGGCGCTCGTAACATTTTAGCGGCGGGGCACGCCGTTCTTGCCTGTGGAGGGATGGTGCAGTCAGGCCGCCCGTCTGAAACAGGAACCAGACGCTGAGCAATCAGTCGTCACCCACTAATGGGAATTCCCCACCTGAGTCACGAAGTGCGAAGGTGGGGGAGGATGTCAA

[0861] SEQ ID NO:125>ISHahl1 gRNA backbone 210nt

[0862] GCGCTGGCACAGCTGCCCTTGCGGCGCGTCCTGTTCACGGGACGAGAACGCCGCTCGGGTGCTGTTGGCTTGGCTGGAGCGGAGTTTATCCGGTCGGGAACCGGCCGATGCGTGGAGGGAGGTCAGACCGGGTCATCCTTTGGATGAATCGGCACTCCCGTCGAAGCGCGAAACTCACGCCATAGCGTAAGCTTGGCGGGAGTAGTTCAT

[0863] SEQ ID NO:126>ISkpn69 gRNA backbone 221nt

[0864] GTCTGCGTGGCGTGTGGATACGAAGCGAACGCCGACATAAACGGCGCTCAAAATATTCTCGCGGCAGGACATGCCGTGTTGTCCGGGATTAACCCCGGACGCGCCCGTAAGGCGGCGTAAGCCGTTAAGCGAGGAACCGGGCAATGCGGTAACGCGTTGCTACCCTACAAAGGGAATCCCCTTCCTGATTCACGTAGTGAGAAGGTGGGGGAGGATGTCAA

[0865] SEQ ID NO:127>ISDge10 gRNA scaffold 207nt

[0866] GTGACCGGGAATGGACGTGCGAATGTGGAGCGGTTCACGACCGCGACCTCAACGCCGCCCGGAACATCAAGCGGGAAGGGCTTTCGCAAATCGTCGTCGCGGGGCACGCGGAGACGTTAAACGCTCGGGGAGAGGGTGTCAGACCTGCGATAGCGGGCAGCCCTCGAtgaAGCGAGAATCCAACGGCTTTAGCCGTTGGAGTGTCAA

[0867] SEQ ID NO:128>ISRor9 gRNA scaffold 205nt

[0868] GTACGTGAATGGCAATGCCCTGTCTGCCTGACTCACCATAATCGCGATATCAATGCGGCGATGAACATCAGGCTGAAGGGCATAACAGAACTAATGGCGGCGGGTCACGTCGTCAAAGCCCACGGAGGCCTGCGTAAGTCCGTTATAACAACGGTTGCGGCCTATGAAGTGGGAAGCCCCGCCCTTtagGGCGGGGAGCAGTCAC

[0869] SEQ ID NO:129>ISSoc3 gRNA scaffold 206nt

[0870] GCATGATTGCCCCCGTTGCGGGTCGTGGGACAGAGACTTGAACGCTGCTATCGAGATTTTGAAGCGAGGACTCAGGGCGGTGGGACTGCCGCTCTCTGGCTGTGGAGGATCCTGGTTTACCAGTCCGTTGAAGCAGCAACTCCGGGAAGTGATTCTCGGAAGCTCCCGTCTACAGCCCGTCAGGGCCTAGCGGGAGAGGATGTCAC

[0871] SEQ ID NO:130>ISKpn85 gRNA backbone 201nt

[0872] GAATGTGGCTACAAAGAGAACGCCGACATAAACGGCGCTCGTAATATTTTAGCGGCAGGACATGCCGTTTTAGCCTGTGAAGTGAACGGTGCATCAATGCCGTCAGCAGCAGGAACCAAGCGACTGAGCAATCAGTTAGCCACCCAGtaaTGGGAATCCCCCACCTGAATCACGTAGTGTGAAGGTGGGGGAGGATGTCAA

[0873] SEQ ID NO:131>ISNsp2 gRNA backbone 196nt

[0874] GAAAACCTAGATTTGTCAATTAGACGCTGGCGTTGTCCTTCATGTCAATCTGAAAATGATCGGGATGGAAACGCTAGTTTGAATATTAAAAGAGTCGGGAGTTCGACTCTAGGCGTAGGAGATGTTAGACAGTCCCAGACTGCAATCTCTGTCtgaTGCCTGAATCCCCCGTTTTCAAAACGGGGGAGTGGCTCAA

[0875] SEQ ID NO:132>ISAba30 gRNA backbone 204ntGCATAGAGTGCGGGTTCGAGGGTAATGCCGATGTGGTCGGCGCGATCAATGTACTAAGGGCGGGGCACGCCCGGTTAGCCTGTGAAGTGAGCGATGCAGTAGGGTCGCCAGCAGCAGGAACCCACCGAAGCGACTCAGACCAGGCTCATTGCCGCGTCtgaGCGCCGTAGGAATCTCCGGCCTTCAGGCCGGGGAGGATGTCAA

[0876] SEQ ID NO:133ISAam1 gRNA backbone 200nt

[0877] GAATGTGGATTCGAGAGCGACAGGGACGTCAATGCGGCAATCAATATCAAACATGAGGGCATGAAACGATTAGCAATAGCCtaaCTTGTCCTCGAACCGTGGGACACACGGGGATCGCTCAGTCAACTTCCCGTCATGAGATGGGATTACCTGAGAAGCCCCCACCTCTAAGCGAAGCGTAGGTGGTGGGAGCATGTCAC

[0878] SEQ ID NO:134>ISYmu1 gRNA backbone 204nt

[0879] GTCAATGCAGCAAGAAATATACTGGCTGAGGGACTCAGGATAAGAGCTCTGACTCCGGGGTCTtagCAATACAGAAGCAAACAGGAACCGCAGGAATTGCGGGGGTAGCTTGGTAAACAAGAGAAACCTCTGCCGGCAAAGAAATAAGCCGGTAAGTATGCTCTGTTCCCAAGAATCTCGTGACTTTAGTCATGAGAGTTTCAA

[0880] SEQ ID NO:135>ISCytsp1 gRNA backbone 201nt

[0881] GATAGAGATTACAATTCAGCACTCAATATCAAAAAAGAAGGCATTCGCTTATTAGCAAGTGCCtaaAGAAATAAGAGATATAAACTCTTGGAACAAGAGGGTTAGCTTGGTCAATTTCGTCAGCTACCAAAAGTGGCGATTACCCAAGAAGCCCCCACTTCAAGCAACCCGCAAGGGTGGTAAGTGGTGGGAGTAGTTCAC

[0882] SEQ ID NO:136 ISCvi1 gRNA backbone 203nt

[0883] GCAAAGCACGATAGAGACACCAACGCAAGCATAACCATCTTAAATAAAGGGCTGTCAATAGCCtgaCATATATAAAATACCGTAGGGCATACGGGAATTTACGCTTGTGGACACTGTGTAAGACGAGCCAACAAGGTAACTTGTTAGGAACGCAGTAGTGGTTGAAGCAAGAATCCCACGACTTTAGTCGTGTGGAGTGTCAA

[0884] SEQ ID NO:137 ISCvi2 gRNA backbone 200nt

[0885] GAATGCCGCTATCAATATCTTACGTGAGGGATTACGACTACTGACTAAAGTAGCCtaaAACTAAATATTACAGTAGGGATGGAATAGCCCAAACTCATACGCCTGTGGACACTGTGTAAGACGAGCCGTTACGTATCATCGTAGCCAGCGCAGTAGTGGTTGAAGCAGGAAGCTCGGTTACTTGTAACCGAGTAGTTCAC

[0886] SEQ ID NO:138 ISAepa1 gRNA backbone 200nt

[0887] GTTCTTGCGGTTTTGTATTAGATCGTGATCACAATTCAGCAATCAATATCAAAAATGAAGGATTACGCATATTAGCAtaaAGAATAATAACTTTCGGAACGAGAGGGTAAGCTCGGTCAATTTCTTATCGTTAGATAGGATTACCCGAGAAGCCCCCACTTCAAGCGACCCGCAAGGGTGGTAAGTGGAGGGTAGTTCAC

[0888] SEQ ID NO:139 ISBth16 gRNA backbone 246nt

[0889] GTCTGAGCGAGTCTATTCTTGTATATGCGGTGTAAATCTCGATAGAGATTATAACGCAGCTATCAATATCAAAAATGAAGCAATACGCCTATTGGCGTTAGTAtagATAGCAATAAAATAACCCTTGGAACAAGGGAGTTAGCTCGGCTGTCTTAGGACGAATTCGTTAGCTACCAAAAGTAACGACTAACCGAGAAGCCCCCACTTCAAACAGTCCGTTAGGACGTTAAGTGGCGGGTAGTTCAC

[0890] SEQ ID NO:140 mRFP coding sequence

[0891] ATGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCCAGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGATGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTCAAGACCACCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAAGACCGACATCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCGCCGAGGGCCGCCACTCCACCGGCGCC

[0892] SEQ ID NO:141 GFP coding sequence

[0893] GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

[0894] SEQ ID NO:142 Target sequence 1 in hDNMT1 of ISTfu1 / ISYmu1

[0895] GGAGGTGAAAAGGCCCTCAT

[0896] SEQ ID NO:143 Target sequence 1 in hTET1 of ISDge10

[0897] GTGCCCAACAGCTTGTATGA

[0898] SEQ ID NO:144 Target sequence 1 in hTET2 of ISDge10

[0899] TATGGAATACCCTGTATGAA

[0900] The target sequence in hHPRT of SEQ ID NO:145ISDge10

[0901] AGCCCCCCTTGAGCACACAG

[0902] Target sequence 2 in hDNMT1 of SEQ ID NO:146 ISAba30

[0903] ATTCTCCAGGAGGAAGAACC

[0904] Target sequence 2 in hTET1 of SEQ ID NO:147 ISAam1

[0905] GTTATCCATAAAAGAATTCC

[0906] Target sequence 2 in hTET2 of SEQ ID NO:148 ISAam1

[0907] GTAATACAATGTTCTTGTCA

[0908] Target sequence in hDNMT3b of SEQ ID NO:149 ISYmu1

[0909] GTTAGGATTAGGTGGGCTCA

[0910] Target sequence 3 in hDNMT1 of SEQ ID NO:150 ISYmu1

[0911] CAGGTCCTCATAGGTCGAGT

[0912] Target sequence 3 in hTET2 of SEQ ID NO:151 ISYmu1

[0913] TCAAGTGCTGTTTCAACACT

[0914] Target sequence in hPGK1 of SEQ ID NO:152 ISYmu1

[0915] ACTCTCTCCTCAATGCCCTC

[0916] SEQ ID NO:153

[0917] GACACCTTAAACGCTCATGGAGGCTATGTCAGACCTGCTTCGGCGGGCAATGGTCTGCGAAGTGAGAATCACGCGACTTTAGTCGTGTGAGGTTCAA

[0918] SEQ ID NO:154

[0919] GTGGCTGCGGGAATCTCAGACACCTTAAACGCTCATGGAGGCTATGTCAGACCTGCTTCGGCGGGCAATGGTCTGCGAAGTGAGAATCACGCGACTTTAGTCGTGTGAGGTTCAA

[0920] SEQ ID NO:155

[0921] GGCGTGAAGCGTTGGTGGCTGCGGGAATCTCAGACACCTTAAACGCTCATGGAGGCTATGTCAGACCTGCTTCGGCGGGCAATGGTCTGCGAAGTGAGAATCACGCGACTTTAGTCGTGTGAGGTTCAA

[0922] SEQ ID NO:156

[0923] GAACGCTGCGCTGAACATTCGGCGTGAAGCGTTGGTGGCTGCGGGAATCTCAGACACCTTAAACGCTCATGGAGGCTATGTCAGACCTGCTTCGGCGGGCAATGGTCTGCGAAGTGAGAATCACGCGACTTTAGTCGTGTGAGGTTCAA

[0924] SEQ ID NO:157

[0925] GTGGGGAAACCCATGACCGAGACGAGAACGCTGCGCTGAACATTCGGCGTGAAGCGTTGGTGGCTGCGGGAATCTCAGACACCTTAAACGCTCATGGAGGCTATGTCAGACCTGCTTCGGCGGGCAATGGTCTGCGAAGTGAGAATCACGCGACTTTAGTCGTGTGAGGTTCAA

[0926] SEQ ID NO:158

[0927] GTCCGTACATGGACTTGCCCGAACTGTGGGGAAACCCATGACCGAGACGAGAACGCTGCGCTGAACATTCGGCGTGAAGCGTTGGTGGCTGCGGGAATCTCAGACACCTTAAACGCTCATGGAGGCTATGTCAGACCTGCTTCGGCGGGCAATGGTCTGCGAAGTGAGAATCACGCGACTTTAGTCGTGTGAGGTTCAA

[0928] SEQ ID NO:159

[0929] GATTCAAGAATCCCGAAGTGAAGAATCTTGCCGTCCGTACATGGACTTGCCCGAACTGTGGGGAAACCCATGACCGAGACGAGAACGCTGCGCTGAACATTCGGCGTGAAGCGTTGGTGGCTGCGGGAATCTCAGACACCTTAAACGCTCATGGAGGCTATGTCAGACCTGCTTCGGCGGGCAATGGTCTGCGAAGTGAGAATCACGCGACTTTAGTCGTGTGAGGTTCAA

[0930] SEQ ID NO:160

[0931] GTTGTGTCATGACTGCGGATTCAAGAATCCCGAAGTGAAGAATCTTGCCGTCCGTACATGGACTTGCCCGAACTGTGGGGAAACCCATGACCGAGACGAGAACGCTGCGCTGAACATTCGGCGTGAAGCGTTGGTGGCTGCGGGAATCTCAGACACCTTAAACGCTCATGGAGGCTATGTCAGACCTGCTTCGGCGGGCAATGGTCTGCGAAGTGAGAATCACGCGACTTTAGTCGTGTGAGGTTCAA

[0932] SEQ ID NO:161

[0933] GCCCCTACTTTCCATCTAGCCAGTTGTGTCATGACTGCGGATTCAAGAATCCCGAAGTGAAGAATCTTGCCGTCCGTACATGGACTTGCCCGAACTGTGGGGAAACCCATGACCGAGACGAGAACGCTGCGCTGAACATTCGGCGTGAAGCGTTGGTGGCTGCGGGAATCTCAGACACCTTAAACGCTCATGGAGGCTATGTCAGACCTGCTTCGGCGGGCAATGGTCTGCGAAGTGAGAATCACGCGACTTTAGTCGTGTGAGGTTCAA

[0934] SEQ ID NO:162

[0935] GCGTGGTACGGGCGACTGGTATCTAAAGTCAGCCCCTACTTTCCATCTAGCCAGTTGTGTCATGACTGCGGATTCAAGAATCCCGAAGTGAAGAATCTTGCCGTCCGTACATGGACTTGCCCGAACTGTGGGGAAACCCATGACCGAGACGAGAACGCTGCGCTGAACATTCGGCGTGAAGCGTTGGTGGCTGCGGGAATCTCAGACACCTTAAACGCTCATGGAGGCTATGTCAGACCTGCTTCGGCGGGCAATGGTCTGCGAAGTGAGAATCACGCGACTTTAGTCGTGTGAGGTTCAA

[0936] SEQ ID NO:163

[0937] GTTGGGCGGTCTGTGAAGCGCCAACCACCCAAGGTAGAGGCAGCGTGAACGCGCCTCAGCCACGGGAATCCCCCGCCTTCAGGCGTGGGGAGGAAGTCAA

[0938] SEQ ID NO:164

[0939] GGGCGTGGAGACCTCGCCGTTGGGCGGTCTGTGAAGCGCCAACCACCCAAGGTAGAGGCAGCGTGAACGCGCCTCAGCCACGGGAATCCCCCGCCTTCAGGCGTGGGGAGGAAGTCAA

[0940] SEQ ID NO:165

[0941] GCCGCCGGGCTGGCGGTGACAGGGCGTGGAGACCTCGCCGTTGGGCGGTCTGTGAAGCGCCAACCACCCAAGGTAGAGGCAGCGTGAACGCGCCTCAGCCACGGGAATCCCCCGCCTTCAGGCGTGGGGAGGAAGTCAA

[0942] SEQ ID NO:166

[0943] GTGAACGCCGCGAAGAACATCCTCGCCGCCGGGCTGGCGGTGACAGGGCGTGGAGACCTCGCCGTTGGGCGGTCTGTGAAGCGCCAACCACCCAAGGTAGAGGCAGCGTGAACGCGCCTCAGCCACGGGAATCCCCCGCCTTCAGGCGTGGGGAGGAAGTCAA

[0944] SEQ ID NO:167

[0945] GCCACCAGGCCAACGCCGACGTGAACGCCGCGAAGAACATCCTCGCCGCCGGGCTGGCGGTGACAGGGCGTGGAGACCTCGCCGTTGGGCGGTCTGTGAAGCGCCAACCACCCAAGGTAGAGGCAGCGTGAACGCGCCTCAGCCACGGGAATCCCCCGCCTTCAGGCGTGGGGAGGAAGTCAA

[0946] SEQ ID NO:168

[0947] GCGGTGCCGCGCCTGCGGCCACCAGGCCAACGCCGACGTGAACGCCGCGAAGAACATCCTCGCCGCCGGGCTGGCGGTGACAGGGCGTGGAGACCTCGCCGTTGGGCGGTCTGTGAAGCGCCAACCACCCAAGGTAGAGGCAGCGTGAACGCGCCTCAGCCACGGGAATCCCCCGCCTTCAGGCGTGGGGAGGAAGTCAA

[0948] SEQ ID NO:169

[0949] GTGAAAGCCAAGCGGAGTTCCGGTGCCGCGCCTGCGGCCACCAGGCCAACGCCGACGTGAACGCCGCGAAGAACATCCTCGCCGCCGGGCTGGCGGTGACAGGGCGTGGAGACCTCGCCGTTGGGCGGTCTGTGAAGCGCCAACCACCCAAGGTAGAGGCAGCGTGAACGCGCCTCAGCCACGGGAATCCCCCGCCTTCAGGCGTGGGGAGGAAGTCAA

[0950] SEQ ID NO:170

[0951] GGCACTGCGCGCCGGACAACCGTGAAAGCCAAGCGGAGTTCCGGTGCCGCGCCTGCGGCCACCAGGCCAACGCCGACGTGAACGCCGCGAAGAACATCCTCGCCGCCGGGCTGGCGGTGACAGGGCGTGGAGACCTCGCCGTTGGGCGGTCTGTGAAGCGCCAACCACCCAAGGTAGAGGCAGCGTGAACGCGCCTCAGCCACGGGAATCCCCCGCCTTCAGGCGTGGGGAGGAAGTCAA

[0952] SEQ ID NO:171

[0953] GCAGACCTGCCATGCCTGCGGGCACTGCGCGCCGGACAACCGTGAAAGCCAAGCGGAGTTCCGGTGCCGCGCCTGCGGCCACCAGGCCAACGCCGACGTGAACGCCGCGAAGAACATCCTCGCCGCCGGGCTGGCGGTGACAGGGCGTGGAGACCTCGCCGTTGGGCGGTCTGTGAAGCGCCAACCACCCAAGGTAGAGGCAGCGTGAACGCGCCTCAGCCACGGGAATCCCCCGCCTTCAGGCGTGGGGAGGAAGTCAA

[0954] SEQ ID NO:172

[0955] GTGCCACCTGCGTTCACATCGCAGACCTGCCATGCCTGCGGGCACTGCGCGCCGGACAACCGTGAAAGCCAAGCGGAGTTCCGGTGCCGCGCCTGCGGCCACCAGGCCAACGCCGACGTGAACGCCGCGAAGAACATCCTCGCCGCCGGGCTGGCGGTGACAGGGCGTGGAGACCTCGCCGTTGGGCGGTCTGTGAAGCGCCAACCACCCAAGGTAGAGGCAGCGTGAACGCGCCTCAGCCACGGGAATCCCCCGCCTTCAGGCGTGGGGAGGAAGTCAA

[0956] SEQ ID NO:173

[0957] GGCTCCCGTATTCTGCGGGTGCCACCTGCGTTCACATCGCAGACCTGCCATGCCTGCGGGCACTGCGCGCCGGACAACCGTGAAAGCCAAGCGGAGTTCCGGTGCCGCGCCTGCGGCCACCAGGCCAACGCCGACGTGAACGCCGCGAAGAACATCCTCGCCGCCGGGCTGGCGGTGACAGGGCGTGGAGACCTCGCCGTTGGGCGGTCTGTGAAGCGCCAACCACCCAAGGTAGAGGCAGCGTGAACGCGCCTCAGCCACGGGAATCCCCCGCCTTCAGGCGTGGGGAGGAAGTCAA

[0958] SEQ ID NO:174

[0959] GCGGAGACGTTAAACGCTCGGGGAGAGGGTGTCAGACCTGCGATAGCGGGCAGCCCTCGAtgaAGCGAGAATCCAACGGCTTTAGCCGTTGGAGTGTCAA

[0960] SEQ ID NO:175

[0961] GCAAATCGTCGTCGCGGGGCACGCGGAGACGTTAAACGCTCGGGGAGAGGGTGTCAGACCTGCGATAGCGGGCAGCCCTCGAtgaAGCGAGAATCCAACGGCTTTAGCCGTTGGAGTGTCAA

[0962] SEQ ID NO:176

[0963] GAACATCAAGCGGGAAGGGCTTTCGCAAATCGTCGTCGCGGGGCACGCGGAGACGTTAAACGCTCGGGGAGAGGGTGTCAGACCTGCGATAGCGGGCAGCCCTCGAtgaAGCGAGAATCCAACGGCTTTAGCCGTTGGAGTGTCAA

[0964] SEQ ID NO:177

[0965] GACCTCAACGCCGCCCGGAACATCAAGCGGGAAGGGCTTTCGCAAATCGTCGTCGCGGGGCACGCGGAGACGTTAAACGCTCGGGGAGAGGGTGTCAGACCTGCGATAGCGGGCAGCCCTCGAtgaAGCGAGAATCCAACGGCTTTAGCCGTTGGAGTGTCAA

[0966] SEQ ID NO:178

[0967] GTGGAGCGGTTCACGACCGCGACCTCAACGCCGCCCGGAACATCAAGCGGGAAGGGCTTTCGCAAATCGTCGTCGCGGGGCACGCGGAGACGTTAAACGCTCGGGGAGAGGGTGTCAGACCTGCGATAGCGGGCAGCCCTCGAtgaAGCGAGAATCCAACGGCTTTAGCCGTTGGAGTGTCAA

[0968] SEQ ID NO:179

[0969] GTGACCGGGAATGGACGTGCGAATGTGGAGCGGTTCACGACCGCGACCTCAACGCCGCCCGGAACATCAAGCGGGAAGGGCTTTCGCAAATCGTCGTCGCGGGGCACGCGGAGACGTTAAACGCTCGGGGAGAGGGTGTCAGACCTGCGATAGCGGGCAGCCCTCGAtgaAGCGAGAATCCAACGGCTTTAGCCGTTGGAGTGTCAA

[0970] SEQ ID NO:180

[0971] GACCTGACCCTGAGTGACCGGGAATGGACGTGCGAATGTGGAGCGGTTCACGACCGCGACCTCAACGCCGCCCGGAACATCAAGCGGGAAGGGCTTTCGCAAATCGTCGTCGCGGGGCACGCGGAGACGTTAAACGCTCGGGGAGAGGGTGTCAGACCTGCGATAGCGGGCAGCCCTCGAtgaAGCGAGAATCCAACGGCTTTAGCCGTTGGAGTGTCAA

[0972] SEQ ID NO:181

[0973] GAATGCGGCAGCATCAACGCAGACCTGACCCTGAGTGACCGGGAATGGACGTGCGAATGTGGAGCGGTTCACGACCGCGACCTCAACGCCGCCCGGAACATCAAGCGGGAAGGGCTTTCGCAAATCGTCGTCGCGGGGCACGCGGAGACGTTAAACGCTCGGGGAGAGGGTGTCAGACCTGCGATAGCGGGCAGCCCTCGAtgaAGCGAGAATCCAACGGCTTTAGCCGTTGGAGTGTCAA

[0974] SEQ ID NO:182

[0975] GTCCAGCAAGCTGTGTGGGGAATGCGGCAGCATCAACGCAGACCTGACCCTGAGTGACCGGGAATGGACGTGCGAATGTGGAGCGGTTCACGACCGCGACCTCAACGCCGCCCGGAACATCAAGCGGGAAGGGCTTTCGCAAATCGTCGTCGCGGGGCACGCGGAGACGTTAAACGCTCGGGGAGAGGGTGTCAGACCTGCGATAGCGGGCAGCCCTCGAtgaAGCGAGAATCCAACGGCTTTAGCCGTTGGAGTGTCAA

[0976] SEQ ID NO:183

[0977] GCGGTCATAGACCGCTGGTTTCCGTCCAGCAAGCTGTGTGGGGAATGCGGCAGCATCAACGCAGACCTGACCCTGAGTGACCGGGAATGGACGTGCGAATGTGGAGCGGTTCACGACCGCGACCTCAACGCCGCCCGGAACATCAAGCGGGAAGGGCTTTCGCAAATCGTCGTCGCGGGGCACGCGGAGACGTTAAACGCTCGGGGAGAGGGTGTCAGACCTGCGATAGCGGGCAGCCCTCGAtgaAGCGAGAATCCAACGGCTTTAGCCGTTGGAGTGTCAA

[0978] SEQ ID NO:184

[0979] GTGGCACCGGAAGTGGCTGGCGGTCATAGACCGCTGGTTTCCGTCCAGCAAGCTGTGTGGGGAATGCGGCAGCATCAACGCAGACCTGACCCTGAGTGACCGGGAATGGACGTGCGAATGTGGAGCGGTTCACGACCGCGACCTCAACGCCGCCCGGAACATCAAGCGGGAAGGGCTTTCGCAAATCGTCGTCGCGGGGCACGCGGAGACGTTAAACGCTCGGGGAGAGGGTGTCAGACCTGCGATAGCGGGCAGCCCTCGAtgaAGCGAGAATCCAACGGCTTTAGCCGTTGGAGTGTCAA

[0980] SEQ ID NO:185

[0981] GTCGCCAGCAGCAGGAACCCACCGAAGCGACTCAGACCAGGCTCATTGCCGCGTCTGAGCGCCGTAGGAATCTCCGGCCTTCAGGCCGGGGAGGATGTCAA

[0982] SEQ ID NO:186

[0983] GAAGTGAGCGATGCAGTAGGGTCGCCAGCAGCAGGAACCCACCGAAGCGACTCAGACCAGGCTCATTGCCGCGTCtgaGCGCCGTAGGAATCTCCGGCCTTCAGGCCGGGGAGGATGTCAA

[0984] SEQ ID NO:187

[0985] GCACGCCCGGTTAGCCTGTGAAGTGAGCGATGCAGTAGGGTCGCCAGCAGCAGGAACCCACCGAAGCGACTCAGACCAGGCTCATTGCCGCGTCtgaGCGCCGTAGGAATCTCCGGCCTTCAGGCCGGGGAGGATGTCAA

[0986] SEQ ID NO:188

[0987] GATCAATGTACTAAGGGCGGGGCACGCCCGGTTAGCCTGTGAAGTGAGCGATGCAGTAGGGTCGCCAGCAGCAGGAACCCACCGAAGCGACTCAGACCAGGCTCATTGCCGCGTCtgaGCGCCGTAGGAATCTCCGGCCTTCAGGCCGGGGAGGATGTCAA

[0988] SEQ ID NO:189

[0989] GTAATGCCGATGTGGTCGGCGCGATCAATGTACTAAGGGCGGGGCACGCCCGGTTAGCCTGTGAAGTGAGCGATGCAGTAGGGTCGCCAGCAGCAGGAACCCACCGAAGCGACTCAGACCAGGCTCATTGCCGCGTCtgaGCGCCGTAGGAATCTCCGGCCTTCAGGCCGGGGAGGATGTCAA

[0990] SEQ ID NO:190

[0991] GCATAGAGTGCGGGTTCGAGGGTAATGCCGATGTGGTCGGCGCGATCAATGTACTAAGGGCGGGGCACGCCCGGTTAGCCTGTGAAGTGAGCGATGCAGTAGGGTCGCCAGCAGCAGGAACCCACCGAAGCGACTCAGACCAGGCTCATTGCCGCGTCtgaGCGCCGTAGGAATCTCCGGCCTTCAGGCCGGGGAGGATGTCAA

[0992] SEQ ID NO:191

[0993] GACACAAGAGCGGTTCGAGTGCATAGAGTGCGGGTTCGAGGGTAATGCCGATGTGGTCGGCGCGATCAATGTACTAAGGGCGGGGCACGCCCGGTTAGCCTGTGAAGTGAGCGATGCAGTAGGGTCGCCAGCAGCAGGAACCCACCGAAGCGACTCAGACCAGGCTCATTGCCGCGTCtgaGCGCCGTAGGAATCTCCGGCCTTCAGGCCGGGGAGGATGTCAA

[0994] SEQ ID NO:192

[0995] GTCGGCGGATAATCGCCAGACACAAGAGCGGTTCGAGTGCATAGAGTGCGGGTTCGAGGGTAATGCCGATGTGGTCGGCGCGATCAATGTACTAAGGGCGGGGCACGCCCGGTTAGCCTGTGAAGTGAGCGATGCAGTAGGGTCGCCAGCAGCAGGAACCCACCGAAGCGACTCAGACCAGGCTCATTGCCGCGTCtgaGCGCCGTAGGAATCTCCGGCCTTCAGGCCGGGGAGGATGTCAA

[0996] SEQ ID NO:193

[0997] GCCCGTGTTGTGCTCATGTGTCGGCGGATAATCGCCAGACACAAGAGCGGTTCGAGTGCATAGAGTGCGGGTTCGAGGGTAATGCCGATGTGGTCGGCGCGATCAATGTACTAAGGGCGGGGCACGCCCGGTTAGCCTGTGAAGTGAGCGATGCAGTAGGGTCGCCAGCAGCAGGAACCCACCGAAGCGACTCAGACCAGGCTCATTGCCGCGTCtgaGCGCCGTAGGAATCTCCGGCCTTCAGGCCGGGGAGGATGTCAA

[0998] SEQ ID NO:194

[0999] GAACACCAGCCGAACCTGCCCGTGTTGTGCTCATGTGTCGGCGGATAATCGCCAGACACAAGAGCGGTTCGAGTGCATAGAGTGCGGGTTCGAGGGTAATGCCGATGTGGTCGGCGCGATCAATGTACTAAGGGCGGGGCACGCCCGGTTAGCCTGTGAAGTGAGCGATGCAGTAGGGTCGCCAGCAGCAGGAACCCACCGAAGCGACTCAGACCAGGCTCATTGCCGCGTCtgaGCGCCGTAGGAATCTCCGGCCTTCAGGCCGGGGAGGATGTCAA

[1000] SEQ ID NO:195

[1001] GGCTCGTTGCCGTGCCGCCTAAGAACACCAGCCGAACCTGCCCGTGTTGTGCTCATGTGTCGGCGGATAATCGCCAGACACAAGAGCGGTTCGAGTGCATAGAGTGCGGGTTCGAGGGTAATGCCGATGTGGTCGGCGCGATCAATGTACTAAGGGCGGGGCACGCCCGGTTAGCCTGTGAAGTGAGCGATGCAGTAGGGTCGCCAGCAGCAGGAACCCACCGAAGCGACTCAGACCAGGCTCATTGCCGCGTCtgaGCGCCGTAGGAATCTCCGGCCTTCAGGCCGGGGAGGATGTCAA

[1002] SEQ ID NO:196

[1003] GGGACACACGGGGATCGCTCAGTCAACTTCCCGTCATGAGATGGGATTACCTGAGAAGCCCCCACCTCTAAGCGAAGCGTAGGTGGTGGGAGCATGTCAC

[1004] SEQ ID NO:197

[1005] GCCtaaCTTGTCCTCGAACCGTGGGACACACGGGGATCGCTCAGTCAACTTCCCGTCATGAGATGGGATTACCTGAGAAGCCCCCACCTCTAAGCGAAGCGTAGGTGGTGGGAGCATGTCAC

[1006] SEQ ID NO:198

[1007] GCATGAAACGATTAGCAATAGCCtaaCTTGTCCTCGAACCGTGGGACACACGGGGATCGCTCAGTCAACTTCCCGTCATGAGATGGGATTACCTGAGAAGCCCCCACCTCTAAGCGAAGCGTAGGTGGTGGGAGCATGTCAC

[1008] SEQ ID NO:199

[1009] GCAATCAATATCAAACATGAGGGCATGAAACGATTAGCAATAGCCtaaCTTGTCCTCGAACCGTGGGACACACGGGGATCGCTCAGTCAACTTCCCGTCATGAGATGGGATTACCTGAGAAGCCCCCACCTCTAAGCGAAGCGTAGGTGGTGGGAGCATGTCAC

[1010] SEQ ID NO:200

[1011] GACAGGGACGTCAATGCGGCAATCAATATCAAACATGAGGGCATGAAACGATTAGCAATAGCCtaaCTTGTCCTCGAACCGTGGGACACACGGGGATCGCTCAGTCAACTTCCCGTCATGAGATGGGATTACCTGAGAAGCCCCCACCTCTAAGCGAAGCGTAGGTGGTGGGAGCATGTCAC

[1012] SEQ ID NO:201

[1013] GAATGTGGATTCGAGAGCGACAGGGACGTCAATGCGGCAATCAATATCAAACATGAGGGCATGAAACGATTAGCAATAGCCtaaCTTGTCCTCGAACCGTGGGACACACGGGGATCGCTCAGTCAACTTCCCGTCATGAGATGGGATTACCTGAGAAGCCCCCACCTCTAAGCGAAGCGTAGGTGGTGGGAGCATGTCAC

[1014] SEQ ID NO:202

[1015] GAGCGTACATTCCGCTGTGAATGTGGATTCGAGAGCGACAGGGACGTCAATGCGGCAATCAATATCAAACATGAGGGCATGAAACGATTAGCAATAGCCtaaCTTGTCCTCGAACCGTGGGACACACGGGGATCGCTCAGTCAACTTCCCGTCATGAGATGGGATTACCTGAGAAGCCCCCACCTCTAAGCGAAGCGTAGGTGGTGGGAGCATGTCAC

[1016] SEQ ID NO:203

[1017] GTAAAGGAATCTTTATCGCTTTCTGAGCGTACATTCCGCTGTGAATGTGGATTCGAGAGCGACAGGGACGTCAATGCGGCAATCAATATCAAACATGAGGGCATGAAACGATTAGCAATAGCCtaaCTTGTCCTCGAACCGTGGGACACACGGGGATCGCTCAGTCAACTTCCCGTCATGAGATGGGATTACCTGAGAAGCCCCCACCTCTAAGCGAAGCGTAGGTGGTGGGAGCATGTCAC

[1018] SEQ ID NO:204

[1019] GTTCATGTTGCGATCAAGTAAAGGAATCTTTATCGCTTTCTGAGCGTACATTCCGCTGTGAATGTGGATTCGAGAGCGACAGGGACGTCAATGCGGCAATCAATATCAAACATGAGGGCATGAAACGATTAGCAATAGCCtaaCTTGTCCTCGAACCGTGGGACACACGGGGATCGCTCAGTCAACTTCCCGTCATGAGATGGGATTACCTGAGAAGCCCCCACCTCTAAGCGAAGCGTAGGTGGTGGGAGCATGTCAC

[1020] SEQ ID NO:205

[1021] GGTTTCCGTCATCTAAAACTTGTTCATGTTGCGATCAAGTAAAGGAATCTTTATCGCTTTCTGAGCGTACATTCCGCTGTGAATGTGGATTCGAGAGCGACAGGGACGTCAATGCGGCAATCAATATCAAACATGAGGGCATGAAACGATTAGCAATAGCCtaaCTTGTCCTCGAACCGTGGGACACACGGGGATCGCTCAGTCAACTTCCCGTCATGAGATGGGATTACCTGAGAAGCCCCCACCTCTAAGCGAAGCGTAGGTGGTGGGAGCATGTCAC

[1022] SEQ ID NO:206

[1023] GCTTATCAAAATAGATAAGTGGTTTCCGTCATCTAAAACTTGTTCATGTTGCGATCAAGTAAAGGAATCTTTATCGCTTTCTGAGCGTACATTCCGCTGTGAATGTGGATTCGAGAGCGACAGGGACGTCAATGCGGCAATCAATATCAA ACATGAGGGCATGAAACGATTAGCAATAGCCtaaCTTGTCCTCGAACCGTGGGACACACGGGGATCGCTCAGTCAACTTCCCGTCATGAGATGGGATTACCTGAGAAGCCCCCACCTCTAAGCGAAGCGTAGGTGGTGGGAGCATGTCAC

[1024] SEQ ID NO:207

[1025] GTAGCTTGGTAAACAAGAGAAACCTCTGCCGGCAAAGAAATAAGCCGGTAAGTATGCTCTGTTCCCAAGAATCTCGTGACTTTAGTCATGAGAGTTTCAA

[1026] SEQ ID NO:208

[1027] GAACCGCAGGAATTGCGGGGGTAGCTTGGTAAACAAGAGAAACCTCTGCCGGCAAAGAAATAAGCCGGTAAGTATGCTCTGTTCCCAAGAATCTCGTGACTTTAGTCATGAGAGTTTCAA

[1028] SEQ ID NO:209

[1029] GCAATACAGAAGCAAACAGGAACCGCAGGAATTGCGGGGGTAGCTTGGTAAACAAGAGAAACCTCTGCCGGCAAAGAAATAAGCCGGTAAGTATGCTCTGTTCCCAAGAATCTCGTGACTTTAGTCATGAGAGTTTCAA

[1030] SEQ ID NO:210

[1031] GAGCTCTGACTCCGGGGTCTtagCATACAGAAGCAAACAGGAACCGCAGGAATTGCGGGGGTAGCTTGGTAAACAAGAGAAACCTCTGCCGGCAAAGAAATAAGCCGGTAAGTATGCTCTGTTCCCAAGAATCTCGTGACTTTAGTCATGAGAGTTTCAA

[1032] SEQ ID NO:211

[1033] GCTGAGGGACTCAGGATAAGAGCTCTGACTCCGGGGTCTtagCAATACAGAAGCAAACAGGAACCGCAGGAATTGCGGGGGTAGCTTGGTAAACAAGAGAAACCTCTGCCGGCAAAGAAATAAGCCGGTAAGTATGCTCTGTTCCCAAGAATCTCGTGACTTTTAGTCATGAGAGTTTCAA

[1034] SEQ ID NO:212

[1035] GTCAATGCAGCAAGAAATATACTGGCTGAGGGACTCAGGATAAGAGCTCTGACTCCGGGGTCTTAGCAATACAGAAGCAAACAGGAACCGCAGGAATTGCGGGGGTAGCTTGGTAAACAAGAGAAACCTCTGCCGGCAAAGAAATAAGCCGGTAAGTATGCTCTGTTCCCAAGAATCTCGTGACTTTAGTCATGAGAGTTTCAA

[1036] SEQ ID NO:213

[1037] GCCCACCATGACCGTGACGTCAATGCAGCAAGAAATATACTGGCTGAGGGACTCAGGATAAGAGCTCTGACTCCGGGGTCTtagCATACAGAAGCAAACAGGAACCGCAGGAATTGCGGGGGTAGCTTGGTAAACAAGAGAAACCTCTGCCGGCAAAGAAATAAGCCGGTAAGTATGCTCTGTTCCCAAGAATCTCGTGACTTTAGTCATGAGAGTTTCAA

[1038] SEQ ID NO:214

[1039] GACCTGTCCTGTTTGTCATGCCCACCATGACCGTGACGTCAATGCAGCAAGAAATATACTGGCTGAGGGACTCAGGATAAGAGCTCTGACTCCGGGGTCTtagCAATACAGAAGCAAACAGGAACCGCAGGAATTGCGGGGGTAGCTTGGTAAACAAGAGAAACCTCTGCCGGCAAAGAAATAAGCCGGTAAGTATGCTCTGTTCCCAAGAATCTCGTGACTTTAGTCATGAGAGTTTCAA

[1040] SEQ ID NO:215

[1041] GCCTCTCCATGTAAGGGAGTGGACCTGTCCTGTTTGTCATGCCCACCATGACCGTGACGTCAATGCAGCAAGAAATATACTGGCTGAGGGACTCAGGATAAGAGCTCTGACTCCGGGGTCTtagCAATACA GAAGCAAACAGGAACCGCAGGAATTGCGGGGGTAGCTTGGTAAACAAGAGAAACCTCTGCCGGCAAAGAAATAAGCCGGTAAGTATGCTCTGTTCCCAAGAATCTCGTGACTTTAGTCATGAGAGTTTCAA

[1042] SEQ ID NO:216

[1043] GCCACAAGGACAGGAAGAAGCCTCTCCATGTAAGGGAGTGGACCTGTCCTGTTTGTCATGCCCACCATGACCGTGACGTCAATGCAGCAAGAAATATACTGGCTGAGGGACTCAGGATAAAGAGCTTCTGACTCCGGGGTCT tagCAATACAGAAGCAAACAGGAACCGCAGGAATTGCGGGGGTAGCTTGGTAAACAAGAGAAACCTCTGCCGGCAAAGAAATAAGCCGGTAAGTATGCTCTGTTCCCAAGAATCTCGTGACTTTAGTCATGAGAGTTTCAA

[1044] SEQ ID NO:217

[1045] GTCAGATATGCTCAGAGTGCGGCCACAAGGACAGGAAGAAGCCTCTCCATGTAAGGGAGTGGACCTGTCCTGTTTGTCATGCCCACCATGACCGTGACGTCAATGCAGCAAGAAATATACTGGCTGAGGGACTCAGGATAAGAGCTCTGACTCCGGGGTCTtagCAATACAGAAGCAAACAGGAACCGCAGGAATTGCGGGGGTAGCTTGGTAAACAAGAGAAACCTCTGCCGGCAAAGAAATAAGCCGGTAAGTATGCTCTGTTCCCAAGAATCTCGTGACTTTAGTCATGAGAGTTTCAA

[1046] SEQ ID NO:218 F-DNMT1-1

[1047] GAGCAGCTGTAGGCCAAGTC

[1048] SEQ ID NO:219 R-DNMT1-1

[1049] GGTAGAGAGGGGGTTTCACC

[1050] SEQ ID NO:220 F-TET1-1

[1051] CTTATTCCTTCCACAGATCGAG

[1052] SEQ ID NO:221 R-TET1-1

[1053] CTGTACTTGAAGGCTTTGGC

[1054] SEQ ID NO:222 F-TET2-1

[1055] GAAGCAAGCCTGATGGAACAGG

[1056] SEQ ID NO:223 R-TET2-1

[1057] GGGCTACAGAACTCACAGATTC

[1058] SEQ ID NO:224 F-HPRT

[1059] CACAGTTCACTCCAGCCTCAAC

[1060] SEQ ID NO:225 R-HPRT

[1061] CTCACTGTAACCAAGTGAAATG

[1062] SEQ ID NO:226 F-DNMT1-2

[1063] AATTCGCGCACCTACTCCAA

[1064] SEQ ID NO:227 R-DNMT1-2

[1065] GGCCTATGCCATTGAACCCT

[1066] SEQ ID NO:228 F-TET1-2

[1067] AGGTAAGCAAACAGTCAAGGGG

[1068] SEQ ID NO:229 R-TET1-2

[1069] AGGGATGCAACTGTGTTCTTT

[1070] SEQ ID NO:230 F-TET2-2

[1071] CGTAACTTCGGGGTAAGCCA

[1072] SEQ ID NO:231 R-TET2-2

[1073] ATCAGCATCACAGGCCTCAC

[1074] SEQ ID NO:232 F-DNMT3b

[1075] CCAACTTGGTTATGGTGCATT

[1076] SEQ ID NO:233 R-DNMT3b

[1077] GGGTGAGTTTTCATTGCCCA

[1078] SEQ ID NO:234 ​​F-DNMT1-3

[1079] TTTCTGACATTTGGGTACGG

[1080] SEQ ID NO:235 R-DNMT1-3

[1081] TACCTCTGTCCCAGCGTGA

[1082] SEQ ID NO:236 F-TET2-3

[1083] GAGGCATCACTGCCATCAAT

[1084] SEQ ID NO:237 R-TET2-3

[1085] GTCTGGCCAAAGAATGATCC

[1086] SEQ ID NO:238 F-PGK1

[1087] CCAAGGTGGCTGCAGCATTTCAC

[1088] SEQ ID NO:239 R-PGK1

[1089] CACTCTACTTTGTTTCAGGCC

[1090] SEQ ID NO:240 is used to screen target sequences for TnpB.

[1091] GCTCGGAGATCATCATTGCG

Claims

1. A recombinant gene editing system comprising: A TnpB polypeptide having RNA-directed endonuclease activity, or a polynucleotide containing a nucleotide sequence encoding a TnpB polypeptide, wherein the amino acid sequence of the TnpB polypeptide is shown in SEQ ID NO: 24, and Guide RNA (gRNA), or a polynucleotide comprising a nucleotide sequence encoding the gRNA, the gRNA comprising a targeting region capable of hybridizing to a nucleotide sequence of interest and a backbone region capable of binding to the TnpB polypeptide, the sequence of the backbone region being shown in any one of SEQ ID NOs: 208-210.

2. The recombinant gene editing system according to claim 1, wherein the TnpB polypeptide comprises a DDE motif, the DDE motif comprising D185, E279 and D361 of SEQ ID NO:

24.

3. The recombinant gene editing system of claim 1, wherein the TnpB polypeptide can recognize a transposon-associated motif (TAM) adjacent to the nucleotide sequence of interest.

4. The recombinant gene editing system according to claim 3, wherein the TnpB polypeptide can recognize a TAM consisting of four consecutive nucleotides.

5. The recombinant gene editing system according to claim 1, wherein the TnpB polypeptide is derived from microorganisms selected from Youngiibacter multivorans.

6. The recombinant gene editing system according to claim 1, comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises a nucleotide sequence encoding a TnpB polypeptide and the second polynucleotide comprises a nucleotide sequence encoding gRNA.

7. The recombinant gene editing system according to claim 1, further comprising heteropolynucleotides.

8. The recombinant gene editing system according to claim 7, wherein the heteropolynucleotide is an expression cassette, a transgene, donor DNA, or a polynucleotide modification template.

9. A recombination system comprising: Modified TnpB peptides, or polynucleotides containing nucleotide sequences encoding modified TnpB peptides, and Guide RNA (gRNA), or a polynucleotide comprising a nucleotide sequence encoding the gRNA, the gRNA comprising a targeting region capable of hybridizing to a nucleotide sequence of interest and a backbone region capable of binding to the TnpB polypeptide, the sequence of the backbone region being shown in any one of SEQ ID NOs: 208-210. in, The TnpB polypeptide contains a DDE motif, and its amino acid sequence is shown in SEQ ID NO:

24. The TnpB polypeptide possesses RNA-directed endonuclease activity. Compared to the TnpB polypeptide shown in SEQ ID NO: 24, the modified TnpB polypeptide contains modifications in the DDE motif, which includes D185, E279 and D361 of SEQ ID NO:

24. The modified TnpB polypeptide has the ability to recognize and bind DNA molecules, but loses the activity of cleaving double-stranded DNA.

10. The recombination system of claim 9, wherein the modification is selected from D185A, E279A and / or D361A compared to SEQ ID NO:

24.

11. The recombinant system of claim 9, comprising a first polynucleotide and a second polynucleotide, the first polynucleotide comprising a nucleotide sequence encoding a modified TnpB polypeptide, and the second polynucleotide comprising a nucleotide sequence encoding a gRNA.

12. The recombinant system according to any one of claims 9-11, wherein the gRNA further comprises one or more protein-binding domains.

13. An isolated polynucleotide comprising a nucleotide sequence encoding the recombinant gene editing system of any one of claims 1-8 and / or the recombinant system of any one of claims 9-12.

14. An architecture comprising the isolated polynucleotide of claim 13.

15. A vector comprising the isolated polynucleotide of claim 13.

16. A recombinant adeno-associated virus (rAAV) whose genome comprises a nucleotide sequence encoding a recombinant gene editing system according to any one of claims 1-8 or a recombinant system according to any one of claims 9-12.

17. The rAAV of claim 16, wherein the genome comprises: A first expression cassette encoding a TnpB peptide or a modified TnpB peptide; and The second expression cassette encoding guide RNA (gRNA).

18. The rAAV of claim 17, wherein the first expression cassette comprises less than 4,700 nucleotides, less than 4,600 nucleotides, less than 4,500 nucleotides, less than 4,400 nucleotides, less than 4,300 nucleotides, less than 4,200 nucleotides, less than 4,100 nucleotides, or less than 4,000 nucleotides.

19. A method for introducing a double-strand break into a polynucleotide of interest for non-diagnostic or therapeutic purposes, comprising the step of contacting the polynucleotide with a recombinant gene editing system according to any one of claims 1-8.

20. A method for modifying a genomic sequence in a cell for non-diagnostic or therapeutic purposes, comprising the step of introducing the recombinant gene editing system of any one of claims 1-8 or the recombinant system of any one of claims 9-12 into the cell.

21. The method of claim 20, wherein the cell is a prokaryotic cell or a eukaryotic cell.

22. Use of the recombinant gene editing system of any one of claims 1-8 or the recombinant system of any one of claims 9-12 in the preparation of a medicament for modifying genomic sequences in cells.

23. Use of the recombinant gene editing system of any one of claims 1-8 or the recombinant system of any one of claims 9-12 in the preparation of reagents for modifying genomic sequences in cells.

24. The use according to claim 22 or 23, wherein the cell is a prokaryotic cell or a eukaryotic cell.

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