TnpB-based genome editor

Abstract

Provided herein are novel TnpB polypeptides having RNA guided endonuclease activity. Also provided herein is a gene editing system comprising the TnpB polypeptide of the invention or a disarmed variant thereof, or a fusion polypeptide comprising the same, and a method of gene editing using the TnpB polypeptide of the invention or a disarmed variant thereof, or the fusion polypeptide or the gene editing system.

Description

Technical Field

[0001] The present invention relates to molecular biology. In particular, the present invention provides a novel RNA-guided system for gene editing. Background Art

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

[0003] Genome-editing technologies such as meganucleases, designer 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 utilize designer nucleases that need to be redesigned for each target site, making them expensive and time-consuming to prepare.

[0004] Recently, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas systems have become popular, which contain different domains of effector proteins covering various activities (DNA recognition, binding, and optional cleavage). However, Cas nucleases are generally large in size, making it difficult to deliver CRISPR-Cas systems into cells.

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

[0006] The insertion sequences of the IS200 / IS605 and IS607 families are among the simplest mobile genetic elements, containing only the genes required for their transposition and its regulation. These elements encode the tnpA transposase, which is required for movement, and usually carry the auxiliary tnpB gene, which is dispensable for transposition. It is reported that the TnpB protein (ISDra2 TnpB) has RNA-guided DNA endonuclease activity (Karvelis et al., Transposon-associated TnpB is aprogrammable RNA-guided DNA endonuclease. Nature, 2021, 559: 692-696). TnpB is the functional ancestor of CRISPR–Cas nucleases and has been established as the prototype of the new genome editing system. The length of the TnpB protein is generally much smaller than that of the Cas protein, making it easier to deliver into cells.

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

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

[0009] To meet these needs, the inventors have identified a number of TnpB polypeptides with RNA-guided DNA endonuclease activity. The identification of TnpB polypeptides increases the possibility of editing various genomic regions that are inaccessible to TnpB polypeptides of the prior art. In addition, the TnpB polypeptides of the present disclosure provide higher editing efficiencies than prior art TnpB polypeptides, even comparable to Cas9 nuclease.

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

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

[0012] - 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 polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding a gRNA,

[0013] wherein the TnpB polypeptide is derived from a microorganism selected from the group consisting of 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 Bacteriophage c-st, Methanosarcina acetic acidophilus, acetivorans, 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-guided endonuclease activity.

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

[0015] - a recombinant TnpB polypeptide or a functional fragment thereof,

[0016] - a target double-stranded DNA comprising a nucleotide sequence of interest and a TAM recognized by said TnpB polypeptide; and

[0017] - a recombinant 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 polypeptide or a functional fragment thereof,

[0018] wherein the TnpB polypeptide is derived from a microorganism selected from the group consisting of Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas intermedius, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc sp. PCC 7120, Clostridium botulinum type C C-Stockholm phage c-st, Methanosarcina acetophilus, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Raoultia ornithinelyticus, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Aerobacillus pallidum, and Bacillus thuringiensis, and wherein the TnpB polypeptide has RNA-guided endonuclease activity.

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

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

[0021] - a guide RNA (gRNA) comprising a targeting 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 comprising a nucleotide sequence encoding a gRNA,

[0022] wherein the TnpB polypeptide is derived from a microorganism selected from the group consisting of Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas intermedius, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc sp. PCC 7120, Clostridium botulinum type C C-Stockholm phage c-st, Methanosarcina acetophilus, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Raoultia ornithinelyticus, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Aerobacillus pallidum, and Bacillus thuringiensis, and wherein the TnpB polypeptide has RNA-guided endonuclease activity.

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

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

[0025] - a guide RNA (gRNA) comprising a targeting region capable of hybridizing to a portion of a genomic sequence and a backbone region capable of binding to a TnpB polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding a gRNA,

[0026] wherein the TnpB polypeptide is derived from a microorganism selected from the group consisting of Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas intermedius, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc sp. PCC 7120, Clostridium botulinum type C C-Stockholm phage c-st, Methanosarcina acetophilus, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Raoultia ornithinelyticus, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Aerobacillus pallidum, and Bacillus thuringiensis, and wherein the TnpB polypeptide has RNA-guided endonuclease activity.

[0027] In a fifth aspect, the present disclosure provides a modified TnpB polypeptide comprising a modification in a DDE motif compared to a parent TnpB polypeptide, wherein the parent polypeptide has RNA-guided endonuclease activity, and wherein the modified TnpB loses the activity of cleaving double-stranded DNA.

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

[0029] - the modified TnpB polypeptide or a functional fragment thereof of the fifth aspect, or a polynucleotide comprising a nucleotide sequence encoding the modified TnpB polypeptide or a functional fragment thereof, and

[0030] - A guide RNA (gRNA), comprising a targeting region capable of hybridizing to a nucleotide sequence of interest and a framework region capable of binding to a TnpB polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding a gRNA.

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

[0032] In an eighth aspect, the present disclosure provides a fusion polypeptide comprising a TnpB polypeptide or a functional fragment thereof or a disarmed TnpB polypeptide fused to a fusion partner, wherein the TnpB polypeptide is derived from a microorganism selected from the group consisting of Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas intermedius, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcuse longatus, Nostoc sp. PCC 7120, Clostridium botulinum type C C-Stockholm phage c-st, Methanosarcina aceticidans, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Raoultella ornithinelyticus, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp.CY-G, Clostridium vitabionis, Aerobacillus pallidum, and Bacillus thuringiensis, wherein the TnpB polypeptide has RNA-guided endonuclease activity, and wherein the disarmed TnpB polypeptide loses the activity of cleaving double-stranded RNA.

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

[0034] - a fusion polypeptide of the present disclosure, or a polynucleotide comprising a nucleotide sequence encoding the fusion polypeptide, and

[0035] - A guide RNA (gRNA), comprising a targeting region capable of hybridizing to a nucleotide sequence of interest and a framework region capable of binding to a TnpB polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding a gRNA.

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

[0037] In an eleventh aspect, the present disclosure provides a method for screening the double-stranded DNA cleavage activity of a TnpB polypeptide, comprising the following steps:

[0038] - providing a candidate TnpB polypeptide from a microorganism;

[0039] - providing a gRNA comprising a targeting region and a framework region, wherein the framework region comprises 100-350 nucleotides before the 3' end of the IS, which naturally comprises a nucleotide sequence encoding a TnpB polypeptide;

[0040] - providing a target DNA comprising a nucleotide sequence that hybridizes with the nucleotide sequence of the targeting region and a TAM recognized by the TnpB polypeptide, wherein the TAM consists of 4 or 5 consecutive nucleotides adjacent to the 5' end of the IS;

[0041] - contacting the TnpB polypeptide with the gRNA and target DNA; and

[0042] - Detection of cleavage on target DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 A map of a pair of plasmids for screening TnpB polypeptides having RNA-guided DNA endonuclease activity is shown, including a test plasmid (encoding TnpB polypeptide and gRNA, Figure 1 A) and a reporter plasmid (containing the target sequence, Figure 1 B).

[0044] Figure 2 Shown are the results of screening for TnpB polypeptides having RNA-guided DNA endonuclease activity.

[0045] Figure 3 The depletion ratios of the TnpB polypeptide and the dTnpB polypeptide (TnpB polypeptide in which the DDE motif is substituted with alanine) are shown.

[0046] Figure 4 Shown is an amino acid sequence alignment of TnpB polypeptides having RNA-guided DNA endonuclease activity.

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

[0048] Figure 6 Shown are the structure and mechanism of the fluorescent reporter system.

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

[0050] Figure 8Shown are flow cytometry results for detecting GFP expression, indicating RNA-directed cleavage by ISTfulTnpB, ISDgelO TnpB, ISAba30 TnpB, ISAam1, and ISYmul TnpB polypeptides in a fluorescent reporter system.

[0051] Figure 9 Shown are the editing efficiencies of ISTfu1 TnpB and ISDra2 TnpB polypeptides using different TAMs in a fluorescent reporter system.

[0052] Figure 10 Shown are the results of mismatch cleavage surveyor assays detecting RNA-directed cleavage of ISTful (Panel A), ISDgelO TnpB (Panel B), ISAba30 TnpB (Panel C), ISAam1 (Panel D), and ISYmu1 (Panel E) TnpB polypeptides in human cells.

[0053] Figure 11 Shown are the effects of backbone design on RNA-directed cleavage of the ISDra2 (Panel A), ISTful (Panel B), ISDgelO (Panel C), ISAba30 (Panel D), ISAam1 (Panel E), and ISYmu1 (Panel F) TnpB polypeptides.

[0054] Figure 12 Shown are the distribution of 25 active TnpB proteins and the 10 conserved residues in ISDra2 TnpB (Panel A), which are marked with asterisks and black lines in the bottom column, superimposed domain structures, and the drastic reduction in endonuclease activity of the TnpB mutant (N to A) (Panel B).

[0055] Figure 13 The editing efficiency of ISAam1, ISYmu1, and ISDra2 at 6 randomly selected endogenous sites in HEK293T cells is shown. Data are shown as mean ± SD, n = 3.

[0056] Figure 14 Figure 2 shows gRNA designs for seven nucleases targeting 10 human genomic loci. The nucleases and corresponding TAMs are color-coded. gRNAs are aligned based on strand position. Using CBLB as an example, the gRNAs for ISAam1 and the three Cas12f variants overlap more than the other three nucleases.

[0057] Figure 15The editing efficiency comparison of 2 kinds of TnpB systems and 5 kinds of Cas nucleases at 10 genomic sites in human HEK293T (Figure A) and HCT116 (Figure B) cells is shown, as well as the editing efficiency comparison of ISAam1 (Figure C) or ISYmu1 (Figure D) relative to 5 kinds of Cas nucleases at 3 genomic sites in HEK293T cells. For Figures A and B, each point represents the average efficiency of 3 biological replicates. The distribution is shown as a box plot, where the box represents the median (center line) and interquartile range (IQR, box limits), and the whiskers are represented from the minimum to the maximum value. For Figures C and D, gRNA design is shown on the left figure, and editing efficiency is shown on the right figure. 7 kinds of nucleases are color-coded. Since it is impossible to design overlapping gRNAs targeting the same position in all 7 kinds of nucleases, two groups of overlapping gRNAs were designed for ISAam1, 3 kinds of Cas12f variants and Nme2-C.NR, and ISAam1 and SaCas9 respectively. ISYmu1 is also in a similar situation.

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

[0059] Figure 17 Shown are the off-target to on-target editing efficiency ratios for the top two predicted off-target sites for each nuclease (n=3, Panel A), as well as the off-target levels quantified by iGUIDE analysis at the MAPK8 locus in HEK293T cells (Panel B, where pie charts show the proportions of on-target and off-target reads, respectively). Detailed Description of the Invention

[0061] 1 Definition

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

[0063] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids and include full-length proteins and fragments thereof.

[0064] Endonucleases are enzymes that cut phosphodiester bonds within polynucleotide chains, including restriction endonucleases that cut DNA at specific sites without damaging bases. Examples of endonucleases include, but are not limited to, restriction endonucleases, meganucleases, TAL effector nucleases (TALENs), zinc finger nucleases, and Cas (CRISPR-associated) effector endonucleases. The present disclosure provides a new RNA-guided TnpB endonuclease.

[0065] As used herein, "nucleic acid" refers to a polynucleotide, including a single-stranded or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases. Nucleic acids can 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-stranded or double-stranded RNA and / or DNA and / or RNA-DNA polymers, optionally containing synthetic, non-natural, or altered nucleotide bases. Nucleotides (usually present in 5'-monophosphate form) are represented by their single-letter names as follows: "A" represents adenosine or deoxyadenosine (representing RNA or DNA, respectively), "C" represents cytosine or deoxycytosine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

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

[0067] The term "selectively hybridizes" refers to the hybridization of a nucleic acid sequence to a specific nucleic acid target sequence, preferably under stringent hybridization conditions, to a detectably greater degree (e.g., at least 2 times background) than hybridization to non-target nucleic acid sequences, and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have at least about 80% sequence identity, or 90% sequence identity, and up to 100% sequence identity (i.e., fully complementary) to each other.

[0068] The term "stringent conditions" or "stringent hybridization conditions" includes conditions under which a probe will selectively hybridize to its target sequence in an in vitro hybridization assay. Stringent conditions are sequence-dependent and will vary in different circumstances. By controlling the stringency of the hybridization and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous probing). Alternatively, the stringency conditions can be adjusted to allow for some mismatches in the sequence, thereby detecting a lower degree of similarity (heterologous probing). Those skilled in the art are aware of various conditions for hybridization, including stringent hybridization conditions and highly stringent hybridization conditions. 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 a donor DNA is a DNA region that has a similar sequence to a given "genomic region" in the genome of a cell or organism. The homologous region can be of any length, as long as it is sufficient to promote homologous recombination at the cleavage target site. For example, the homology region can comprise 5-3000 bases or more, e.g., 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 a corresponding genomic region.

[0070] As used herein, a "genomic region" is a segment of a cell's chromosomal or organelle DNA that exists upstream or downstream of a target site, or that also encompasses a portion of the target site (at the 5' or 3' end). 00, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 bases in length.

[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 proportions of homologous and nonhomologous recombination vary between organisms. In general, the length of the homologous region influences 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 Cell Biol 12: 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 the context of nucleotide or amino acid sequences means that the nucleotide bases or amino acid residues in two sequences are the same when aligned for maximum correspondence over a specified comparison window.

[0073] The term "percentage of sequence identity" refers to a value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the nucleotide or amino acid sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) to achieve optimal alignment of the two sequences. The percentage of sequence identity is calculated by dividing the number of matching positions (i.e., positions where the nucleotide bases or amino acid residues are the same in the two sequences) by the total number of positions in the comparison window and multiplying the result by 100. For example, when aligning two sequences, if 950 positions in the two sequences that are optimally aligned are the same over a comparison window of 1000 positions, the sequences are 95% identical to each other.

[0074] A variety of comparison methods have been designed for sequence alignment and calculation of percent identity or similarity, 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 is understood that when sequence analysis software is used for analysis, unless otherwise specified, the analysis results will be based on the "default values" of the referenced program. As used herein, "default values" refers to any set of values ​​or parameters that are initially loaded with the software when it is first initialized.

[0075] "BLAST" is a search algorithm provided by NCBI for finding regions of similarity between biological sequences. The program compares nucleotide or protein sequences to a database of sequences and calculates the statistical significance of the matches to identify sequences that have sufficient similarity to the query sequence so that the similarity would not be expected to arise by chance. BLAST reports the identified sequences and their local alignment with the query sequence. It is well understood by those skilled in the art that many levels of sequence identity can be used to identify polypeptides from other species or natural or synthetic modifications, where such polypeptides have the same or similar function or activity. Useful examples of percentages of identity include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any percentage from 50% to 100%. Indeed, any amino acid identity from 50% to 100% can be used to describe the present disclosure, for example, 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 "centimorgan" (cM) or "mapping unit" is the distance between two polynucleotide sequences, linked genes, markers, target sites, genomes, or any pair thereof, at which 1% of the meiotic products are recombinant. Thus, one centimorgan is equivalent to a distance between two linked genes, markers, target sites, loci, or any pair thereof that is equal to 1% average recombination frequency.

[0077] "Isolated" polynucleotides, polypeptides or proteins are substantially or essentially free of components that are typically found in their naturally occurring environment that accompany or interact with the polynucleotides, polypeptides or proteins. Thus, isolated polynucleotides or polypeptides or proteins are substantially free of other cellular material or culture medium when prepared by recombinant techniques, or are substantially free of chemical precursors or other chemical substances when chemically synthesized. Preferably, "isolated" polynucleotides do not contain sequences that naturally flank the polynucleotides in the genomic DNA of the organism from which the polynucleotides are derived (i.e., sequences at the 5' and 3' ends of the polynucleotides). Isolated polynucleotides and polypeptides can be purified from their naturally occurring cells. Methods for separating or purifying polynucleotides or polypeptides are well known to those skilled in the art. The term also encompasses recombinant or chemically synthesized polynucleotides and polypeptides.

[0078] The term "fragment" refers to a group of consecutive nucleotides or amino acids. In one embodiment, a 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 one embodiment, a 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. A fragment may or may not exhibit the function of a sequence having a certain percentage of identity over the length of the fragment.

[0079] The term "functional fragment" refers to a portion of an isolated polynucleotide or polypeptide that exhibits the same activity or function as the longer or full-length sequence from which it is derived.

[0080] The term "gene" includes a nucleic acid fragment that expresses a functional molecule, such as but not limited to a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.

[0081] The term "endogenous" refers to a sequence or other molecule that occurs naturally in a cell or organism. An endogenous polynucleotide is typically present in the genome of a cell; ie, it is 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 differences in taxonomic derivation (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 is introduced into a plant or animal cell), or differences in sequence (e.g., a polynucleotide obtained from species A, isolated, modified from, and reintroduced into a plant of species A).

[0083] "Allele" is one of several alternative forms of a gene occupying a given locus on a chromosome. When all alleles present at a given locus on a chromosome in a cell or organism are identical, the cell or organism is homozygous at that locus. If the alleles present at a given locus on a chromosome are different, the cell or organism is heterozygous at that locus.

[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 sequences), within, or downstream (3' non-coding sequences) of a coding sequence that influences the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, promoters, translation leader 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 that has been altered by human intervention. The sequence of a "mutated gene" differs from the sequence of the corresponding non-mutated gene by the addition, deletion, insertion, or substitution of at least one nucleotide. In the present disclosure, a mutated gene comprises an alteration induced by the guide polynucleotide / TnpB endonuclease system disclosed herein. A mutant organism is an organism that contains a mutated gene.

[0086] As used herein, a "targeted mutation" is a genetic mutation generated in a target sequence within a gene using any method known to those skilled in the art, including the methods disclosed herein involving directing the TnpB endonuclease system.

[0087] The term "knockout" refers to a DNA sequence in a cell that has been partially or completely disabled, for example, by targeting of a TnpB protein of the present disclosure; for example, the DNA sequence before knockout may encode an amino acid sequence, or may have a regulatory function (eg, a promoter).

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

[0089] The term "domain" refers to a stretch of consecutive nucleotides (which may be RNA, DNA and / or RNA-DNA combination sequences) or consecutive or discontinuous amino acids.

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

[0091] A "codon-optimized" nucleotide sequence is a nucleotide sequence whose codon usage frequency is designed to mimic the preferred codon usage frequency of a host cell. "Optimized" polynucleotides include nucleotide sequences that are optimized for improved expression in a specific heterologous host cell.

[0092] A "promoter" is a nucleotide sequence that is involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. Promoters can be derived entirely from a natural gene, or be composed of different elements derived from different promoters found in nature, and / or comprise synthetic DNA fragments. Those skilled in the art will appreciate that different promoters can direct the expression of a gene in different tissues or cell types, or at different developmental stages, or under different environmental conditions. It is further recognized that because the exact boundaries of regulatory sequences have not been completely defined in most cases, certain variant DNA fragments may have the same promoter activity.

[0093] A promoter that causes a gene to be expressed in most tissues or cell types most of the time is generally referred to as a "constitutive promoter." The term "inducible promoter" or "regulatable promoter" refers to a promoter that selectively expresses a coding sequence or functional RNA in response to the presence of an endogenous or exogenous stimulus, such as a compound (chemical inducer), or in response to an environmental, hormonal, chemical, and / or developmental signal. Inducible or regulatable promoters include, for example, promoters that are induced or regulated by light, heat, stress, flooding or drought, salt stress, osmotic stress, plant hormones, injury, 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 and can be an intrinsic element of the promoter or a heterologous element inserted to enhance promoter activity or tissue specificity.

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

[0096] The term "3' non-coding sequence" is interchangeable with "transcription terminator" or "termination sequence" and refers to nucleotide sequences located downstream of the coding sequence. These include polyadenylation signal sequences and other sequences encoding regulatory signals that affect mRNA processing or gene expression. The polyadenylation signal is typically characterized by the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[0097] The term "RNA transcript" refers to the product produced by the transcription of a DNA sequence catalyzed by RNA polymerase. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is called a primary transcript or pre-mRNA. The RNA transcript derived from the post-transcriptional processing of the pre-mRNA is 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 to 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 in cells or in vitro. "Antisense RNA" refers to RNA transcripts that are complementary to all or part of a target primary transcript or mRNA and can block the expression of a target gene. The complementarity of antisense RNA can be to any part of a specific gene transcript, i.e., in the 5' non-coding region, 3' non-coding region, introns, or coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but has an effect on cellular processes. The terms "complementary" and "reverse complement" are used interchangeably herein with respect to mRNA transcripts and are intended to define the antisense RNA of the message.

[0098] The term "operably linked" refers to the association of nucleotide sequences on individual nucleic acid segments such that the function of one is regulated by the other. For example, a promoter is operably linked to a coding sequence when the promoter is capable of regulating the expression of the coding sequence (i.e., the coding sequence is transcribed under the control of the promoter). A coding sequence can be operably linked to a regulatory sequence in either sense or antisense orientation.

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

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

[0101] The terms "plasmid" and "vector" refer to a linear or circular extrachromosomal element that typically carries genes not involved in the central metabolism of the cell and is usually in the form of double-stranded DNA. Such elements can be autonomously replicating sequences derived from single-stranded or double-stranded DNA or RNA from any source, genome-integrating sequences, bacteriophages, or nucleotide sequences, in linear or circular form, in which multiple nucleotide sequences have been joined or recombined into a unique structure capable of introducing the polynucleotide of interest into the cell.

[0102] When referring to nucleic acid molecules, the term "construct" includes artificial combinations of nucleic acid sequences, for example, regulatory sequences and coding sequences that are not all present simultaneously in nature. When a nucleic acid construct contains the control sequence required for expressing a coding sequence of the present invention, the term is synonymous with the term "expression cassette". For example, a nucleic acid construct can include regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that present in nature. Such a construct can be used alone or in combination with a vector. If a vector is used, the selection of the vector depends on the method by which the vector is introduced into a host cell, which is well known to those skilled in the art. A vector for expressing a coding sequence (for example, comprising an expression construct) is referred to as an "expression vector".

[0103] As used herein, the term "expression" refers to the production of a functional end-product (eg, mRNA, guide RNA, or protein) in precursor or mature form.

[0104] A "mature" protein refers to a post-translationally processed polypeptide (ie, a polypeptide from which any propeptides or precursor peptides present in the primary translation product have been removed).

[0105] "Precursor" protein refers to the primary product of mRNA translation (ie, pre- and pre-peptides are still present). Pre- and pre-peptides can be, but are not limited to, intracellular localization signals.

[0106] As used herein, an "effector" or "effector protein" is a protein that has an activity that includes recognition, binding, and / or cleavage or cleavage of a polynucleotide target. An effector or effector protein can also be an endonuclease, such as the TnpB polypeptide of the present invention. The "effector complex" of the gene editing system includes the gRNA and the TnpB polypeptide that participates in target recognition and binding.

[0107] A "functional fragment" of a TnpB endonuclease refers to a portion of a TnpB endonuclease disclosed herein that retains the ability to recognize, bind and / or cleave (introduce double-strand breaks) a target site. A "functional variant" of a TnpB endonuclease refers to a variant of a TnpB endonuclease disclosed herein that retains the ability to recognize, bind and / or cleave a target sequence.

[0108] TnpB endonucleases may also include multifunctional TnpB endonucleases, which refer to a single polypeptide having endonuclease activity (comprising at least one protein domain that can act as a nuclease) and at least one other function, such as but not limited to the function of forming a complex (comprising 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, such as the TnpB endonuclease described herein, and enable the Cas endonuclease to recognize, optionally bind, and optionally cleave a DNA target site. The guide polynucleotide can be a guide RNA, a guide DNA sequence, or a combination thereof (RNA-DNA combination molecule). For a TnpB polypeptide, the guide RNA is also referred to as a "right element RNA" or "reRNA."

[0110] A "functional fragment" of a guide polynucleotide refers to a portion or subsequence of a guide polynucleotide of the present disclosure that retains the ability to function as a guide polynucleotide. A "functional variant" of a guide polynucleotide refers to a variant of a guide polynucleotide of the present disclosure that retains the ability to function as a guide polynucleotide.

[0111] The terms "targeting domain" and "targeting region" are used interchangeably herein and include a nucleotide sequence that can hybridize (be complementary) to one strand (nucleotide sequence) of a double-stranded DNA target site. The percent complementarity between the targeted 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 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 length. In some embodiments, the variable targeting domain comprises a continuous segment of 12-30 nucleotides. The targeting domain can be composed 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 comprises a nucleotide sequence that interacts with a TnpB polypeptide.

[0113] The term "gRNA / TnpB complex" refers to an RNA component and a TnpB endonuclease that are capable of forming a complex, wherein the complex can guide the TnpB endonuclease to a DNA target site, enabling the TnpB endonuclease to recognize, bind and / or cut (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 chromosomes, chloroplasts, mitochondrial DNA, plasmid DNA) on a chromosome, episome, locus, or any other DNA molecule in the cell genome on which the gRNA / TnpB complex can recognize, bind, and optionally crack or cut. The target site can be an endogenous site in the cell genome, or the target site can be heterologous to the cell so as not to naturally occur in the cell genome, or the target site can be found at a heterologous genomic location compared to its naturally occurring position.

[0115] "Transposon-associated motif" herein 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 not be able to successfully recognize the target DNA sequence. The sequence and length of TAM herein can vary depending on the TnpB protein. The TAM sequence is typically 4 or 5 nucleotides long.

[0116] A "modified" TnpB polypeptide / endonuclease refers to a TnpB polypeptide that comprises at least one amino acid substitution, deletion, insertion, or addition compared to an original or wild-type TnpB polypeptide. If the modified TnpB loses the activity of cleaving DNA molecules but retains the ability to recognize and bind polynucleotides, the modified TnpB polypeptide can be referred to as a "disarmed" TnpB polypeptide.

[0117] "Altered target site," "altered target sequence," "modified target site," and "modified target sequence" are used interchangeably herein to refer to a target sequence as disclosed herein that comprises at least one alteration compared to the unaltered target 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 alteration of at least one nucleotide, or (v) any combination of (i)-(iv).

[0118] "Modified nucleotides" or "edited nucleotides" refer to a nucleotide sequence of interest that comprises at least one change compared to its unmodified nucleotide sequence. Such "changes" 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 alteration of at least one nucleotide, or (v) any combination of (i)-(iv).

[0119] A method of "modifying a target site" and "altering a target site" are used interchangeably herein to refer to a method that produces an altered target site.

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

[0121] The term "polynucleotide modification template" includes a polynucleotide comprising at least one nucleotide modification compared to the nucleotide sequence to be edited. The nucleotide modification can be a substitution, addition, insertion or deletion of at least one nucleotide. Optionally, the polynucleotide modification template may further comprise 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, with respect to sequence position, the term "before" refers to a sequence that appears upstream of another sequence (at the 5' end for nucleotide sequences and at the N-terminus for amino acid sequences). With respect to sequence position, the term "after" refers to a sequence that appears downstream of another sequence (at the 3' end for nucleotide sequences and at the C-terminus for amino acid sequences).

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

[0124] The inventors have identified a number of novel RNA-guided endonucleases, which are TnpB polypeptides encoded by the TnpB gene within the insertion sequence (IS). In other words, TnpB polypeptides can serve as effector proteins in gene editing systems. Sequence analysis revealed that the active TnpB polypeptide contains an N-terminal helix-turn-helix (HTH) domain, a central domain, a C-terminal zinc finger domain, and a DDE motif.

[0125] Therefore, the present disclosure provides an isolated TnpB polypeptide or a functional fragment thereof having RNA-guided 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 a microorganism selected from the group consisting of Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas intermedius, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc sp. PCC7120, Clostridium botulinum type C C-Stockholm phage c-st, Methanosarcina acetivora, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Raoultella ornithinolyticus, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Aerobacillus pallidum, and Bacillus thuringiensis.

[0127] In some embodiments, the TnpB polypeptide is encoded by a tnpB gene in an insertion sequence (IS) selected from the group consisting of ISEfa4, ISAs26, ISCpe2, ISMma22, ISBce3, ISAeme8, ISTfu1, ISCco1, ISSoc3, ISTel2, ISNsp3, ISCbt1, ISMac7, ISEc46, ISSen6, ISHah11, 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 consists 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, insertions, deletions, and / or additions. In some embodiments, the variants are conserved at 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, 350 or more consecutive amino acids 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 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, 350 or more consecutive amino acids 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.

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

[0132] In some embodiments, a TnpB polypeptide of the present disclosure may recognize a TAM of CCAT, CTAC, TGAC, TGAT, TTAC, TTAG, TTAA, TTAT, ACAT, TTTAT, TTTAA, or TTGAT.

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

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

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

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

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

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

[0139] In some embodiments, the TnpB polypeptide or functional fragment thereof disclosed herein is capable of RNA-guided 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, 18, 21, 23 or 24. In some embodiments, the TnpB polypeptide is a functional variant consisting of an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, 18, 21, 23 or 24. The variant may differ from SEQ ID NO:7, 18, 21, 23 or 24 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, insertions, deletions and / or additions. In some embodiments, the variant is conserved at positions corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354 and D361 of SEQ ID NO:7.

[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, 350 or more consecutive amino acids of 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, 350 or more consecutive amino acids of 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 have discovered that the TnpB endonuclease can be disarmed by modifying the DDE motif. Therefore, the present disclosure also provides a modified / disarmed 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 amino acid or a basic amino acid. In some embodiments, at least one amino acid in the motif is substituted with alanine. In some embodiments, the modified TnpB polypeptide comprises

[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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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 positions corresponding to N31, G179, L267, C332, C335, C351, and C354 of SEQ ID NO: 7.

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

[0204] In some embodiments, the modified TnpB polypeptide comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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 positions corresponding to G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO: 7.

[0205] In some embodiments, the modified TnpB polypeptide has the ability to recognize and bind to DNA molecules, but loses the ability to cleave DNA molecules such as double-stranded DNA, ie, is a disarmed TnpB polypeptide.

[0206] 3. Fusion peptide

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

[0208] In some embodiments, the fusion partner is a polypeptide that provides an activity that indirectly increases transcription by acting directly on the target DNA or on a polypeptide associated with the target DNA (e.g., a histone or other DNA binding protein).

[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, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity.

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

[0211] In some embodiments, the fusion partner is a polypeptide that directs single or multiple base editing in a polynucleotide sequence, such as a site-specific deaminase that can change 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 comprise, for example, an active (double-strand break generation), partially active (nicking enzyme), or inactive (unable to cut) TnpB endonuclease and a deaminase (e.g., but not limited to, cytidine deaminase, adenine deaminase, APOBEC1, APOBEC3A, BE2, BE3, BE4, ABE, etc.). In some embodiments, the fusion partner comprises a base editing repair inhibitor and a glycosylase inhibitor (e.g., a uracil glycosylase inhibitor (to prevent uracil removal)).

[0213] In some embodiments, the fusion partner can be a Cas endonuclease or another TnpB endonuclease as described in the present disclosure.

[0214] TnpB polypeptide of the present disclosure, its functional fragment or modification / disarmed TnpB can also be fused with heterologous nuclear localization sequence (NLS).Heterologous NLS herein can have enough intensity to drive TnpB polypeptide, its functional fragment, modification / disarmed TnpB polypeptide or fusion polypeptide in eukaryotic nucleus with detectable amount accumulation.NLS can comprise one (single portion) or multiple (for example, two portions) alkaline, positively charged residues (for example, lysine and / or arginine) short sequence (for example, 2-20 residues).For example, NLS can be operably connected to the N-terminal or C-terminal of TnpB polypeptide.Two or more NLS sequences can be connected to TnpB polypeptide, for example, at the N-terminal or C-terminal of TnpB polypeptide.

[0215] 4. Guide polynucleotides

[0216] The guide polynucleotide can enable the TnpB polypeptide to recognize, bind, and optionally cleave the target. The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence). Optionally, the guide polynucleotide can include at least one nucleotide, a phosphodiester bond, or a linkage modification, such as, but not limited to, locked nucleic acid (LNA), 5-methyl dC, 2,6-diaminopurine, 2'-fluoro A, 2'-fluoro U, 2'-O-methyl RNA, a phosphorothioate bond, a connection to a cholesterol molecule, a connection to a polyethylene glycol molecule, a connection to a spacer 18 (hexaethylene glycol chain) molecule, or a 5' to 3' covalent connection that results in cyclization. A guide polynucleotide comprising only ribonucleic acid is also referred to as a "guide RNA" or "gRNA." The guide polynucleotide can be engineered or synthetic. The gRNA of the TnpB polypeptide is also referred to as a "right element RNA" or "reRNA."

[0217] Guide polynucleotides include chimeric non-naturally occurring guide RNAs that contain regions that do not coexist in nature (i.e., they are heterologous to each other). For example, the chimeric non-naturally occurring guide RNA comprises a targeting region that can hybridize to 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 polypeptide 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, 340 nucleotides before the right end (RE) of the IS from which TnpB is derived. When part or all of one or more stem structures in the backbone are removed, the guide polynucleotide can affect target recognition, binding, and optionally cleavage of the TnpB polypeptide.

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

[0220] In some embodiments, the targeting region and the framework region are selected from the group consisting of a DNA sequence, an RNA sequence, and combinations thereof.

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

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

[0223] The TnpB endonucleases, functional fragments thereof, disarmed TnpB polypeptides and fusion polypeptides disclosed herein 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 polypeptides and fusion polypeptides can be produced using a cell-free protein expression system, or produced synthetically.

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

[0225] The TnpB polypeptide, functional fragment thereof, disarmed TnpB polypeptide and fusion polypeptide, and guide polynucleotide can be expressed in cells, including but not limited to human, non-human, animal, bacterial, fungal, insect, yeast, and plant cells.

[0226] Standard recombinant DNA and molecular cloning techniques used herein 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). Transformation methods are well known to those skilled in the art and are described below.

[0227] Also provided are vectors and constructs, including circular plasmids and linear polynucleotides, comprising a polynucleotide of interest and, optionally, other components, including linkers, adapters, and regulatory sequences.

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

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

[0230] In some embodiments, expression of the TnpB polypeptide and / or guide polynucleotide is driven by a constitutive promoter, an inducible promoter, or a spatiotemporally specific promoter.

[0231] 6. Gene Editing of TnpB Peptide

[0232] 6.1. Recombinant gene editing system

[0233] The present disclosure provides a recombinant gene editing system comprising the novel TnpB polypeptide having RNA-guided endonuclease activity of the present disclosure.

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

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

[0236] - A guide polynucleotide, such as a guide RNA (gRNA), comprising a targeting region capable of hybridizing to a nucleotide sequence of interest and a framework region capable of binding to a TnpB polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding a 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 a microorganism selected from the group consisting of Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas intermedius, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc sp. PCC7120, Clostridium botulinum type C C-Stockholm phage c-st, Methanosarcina acetivora, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Raoultella ornithinolyticus, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Aerobacillus pallidum, and Bacillus thuringiensis.

[0239] In some embodiments, the TnpB polypeptide is encoded by a tnpB gene in an insertion sequence (IS) selected from the group consisting of ISEfa4, ISAs26, ISCpe2, ISMma22, ISBce3, ISAeme8, ISTfu1, ISCco1, ISSoc3, ISTel2, ISNsp3, ISCbt1, ISMac7, ISEc46, ISSen6, ISHah11, 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 consists 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, insertions, deletions, and / or additions. In some embodiments, the variants are conserved at positions corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO: 7.

[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, 350 or more consecutive amino acids 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 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, 350 or more consecutive amino acids 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.

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

[0244] In some embodiments, a TnpB polypeptide of the present disclosure may recognize a TAM of CCAT, CTAC, TGAC, TGAT, TTAC, TTAG, TTAA, TTAT, ACAT, TTTAT, TTTAA, or TTGAT.

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

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

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

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

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

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

[0251] In some embodiments, the TnpB polypeptide or functional fragment thereof disclosed herein is capable of RNA-guided 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, 18, 21, 23 or 24. In some embodiments, the TnpB polypeptide is a functional variant consisting of an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, 18, 21, 23 or 24. The variant may differ from SEQ ID NO:7, 18, 21, 23 or 24 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, insertions, deletions and / or additions. In some embodiments, the variant is conserved at positions corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354 and D361 of SEQ ID NO:7.

[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, 350 or more consecutive amino acids of 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, 350 or more consecutive amino acids of SEQ ID NO: 7, 18, 21, 23 or 24.

[0254] In some embodiments, the TnpB comprises 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 comprises a nucleotide sequence encoding a TnpB polypeptide or a functional fragment thereof, and the second polynucleotide comprises a nucleotide sequence encoding a gRNA.

[0285] In some embodiments, the recombinant gene editing system further comprises a heterologous polynucleotide, such as an expression cassette, a transgene, a donor DNA, or a polynucleotide modification template.

[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, 340 nucleotides preceding the right end (RE) of the IS from which TnpB is derived. In some embodiments, the backbone is modified by removing a portion or all of one or more stem structures in the backbone. In some embodiments, a portion or all of the first stem structure starting from the 3' end is removed.

[0287] In some embodiments, the guide polynucleotide comprises an additional nucleotide sequence at the 5' end of the backbone. In some embodiments, the additional nucleotide sequence can recognize and / or bind to an additional nuclease, such as a TnpB polypeptide or a Cas nuclease of the present disclosure.

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

[0289] 6.2. Compositions, Complexes, and Isolated Cells

[0290] During the gene editing process, a complex or composition can be formed. Therefore, the present disclosure provides a composition or complex comprising

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

[0292] - a target double-stranded DNA comprising a nucleotide sequence of interest and a TAM recognized by the TnpB polypeptide; and

[0293] - A recombinant guide RNA (gRNA) comprising a targeting region capable of hybridizing to a nucleotide sequence of interest and a framework region capable of binding to a TnpB polypeptide or a functional fragment thereof.

[0294] The present disclosure also provides an isolated cell comprising

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

[0296] - a target double-stranded DNA comprising a nucleotide sequence of interest and a TAM recognized by the TnpB polypeptide; and

[0297] - A recombinant guide RNA (gRNA) comprising a targeting region capable of hybridizing to a nucleotide sequence of interest and a framework region capable of binding to a TnpB polypeptide 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 a microorganism selected from the group consisting of Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas intermedius, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc sp. PCC7120, Clostridium botulinum type C C-Stockholm phage c-st, Methanosarcina acetivora, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Raoultella ornithinolyticus, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Aerobacillus pallidum, and Bacillus thuringiensis.

[0300] In some embodiments, the TnpB polypeptide is encoded by a tnpB gene in an insertion sequence (IS) selected from the group consisting of ISEfa4, ISAs26, ISCpe2, ISMma22, ISBce3, ISAeme8, ISTfu1, ISCco1, ISSoc3, ISTel2, ISNsp3, ISCbt1, ISMac7, ISEc46, ISSen6, ISHah11, 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 consists 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, insertions, deletions, and / or additions. In some embodiments, the variants are conserved at positions corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO: 7.

[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, 350 or more consecutive amino acids 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 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, 350 or more consecutive amino acids 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.

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

[0305] In some embodiments, a TnpB polypeptide of the present disclosure may recognize a TAM of CCAT, CTAC, TGAC, TGAT, TTAC, TTAG, TTAA, TTAT, ACAT, TTTAT, TTTAA, or TTGAT.

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

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

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

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

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

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

[0312] In some embodiments, the TnpB polypeptide or functional fragment thereof disclosed herein is capable of RNA-guided 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, 18, 21, 23 or 24. In some embodiments, the TnpB polypeptide is a functional variant consisting of an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, 18, 21, 23 or 24. The variant may differ from SEQ ID NO:7, 18, 21, 23 or 24 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, insertions, deletions and / or additions. In some embodiments, the variant is conserved at positions corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354 and D361 of SEQ ID NO:7.

[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, 350 or more consecutive amino acids of 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, 350 or more consecutive amino acids of SEQ ID NO: 7, 18, 21, 23 or 24.

[0315] In some embodiments, the TnpB comprises 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 embodiments, the recombinant gene editing system comprises a first polynucleotide and a second polynucleotide, the first polynucleotide comprises a nucleotide sequence encoding a TnpB polypeptide or a functional fragment thereof, and the second polynucleotide comprises a nucleotide sequence encoding a gRNA.

[0346] In some embodiments, the composition further comprises a heterologous polynucleotide, such as an expression cassette, a transgene, donor DNA, or a polynucleotide modification template.

[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, 340 nucleotides preceding the right end (RE) of the IS from which TnpB is derived. In some embodiments, the backbone is modified by removing a portion or all of one or more stem structures in the backbone. In some embodiments, a portion or all of the first stem structure starting from the 3' end is removed.

[0348] In some embodiments, the guide polynucleotide comprises an additional nucleotide sequence at the 5' end of the backbone. In some embodiments, the additional nucleotide sequence can recognize and / or bind to an additional nuclease, such as a TnpB polypeptide or a Cas nuclease of the present disclosure.

[0349] In some embodiments, the gRNA further comprises one or more additional protein binding domains. In some embodiments, the composition of isolated cells comprises one or more additional effector polypeptides capable of binding to one or more additional protein binding domains, or a polynucleotide comprising a nucleotide sequence encoding one or more effector polypeptides, to form one or more ribonucleoproteins in tandem.

[0350] 6.3. Disarming the system

[0351] The present disclosure provides a recombination system comprising:

[0352] - a modified TnpB polypeptide of the present disclosure comprising a modification in a DDE motif compared to a parent TnpB polypeptide, or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding the modified TnpB polypeptide or a functional fragment thereof, wherein the parent polypeptide has RNA-guided endonuclease activity and the modified TnpB polypeptide loses the activity of cleaving double-stranded DNA, and

[0353] - A guide RNA (gRNA), comprising a targeting region capable of hybridizing to a nucleotide sequence of interest and a framework region capable of binding to a TnpB polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding a 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 amino acid or a basic amino acid. In some embodiments, at least one amino acid in the motif is substituted with alanine. In some embodiments, the modified TnpB polypeptide comprises

[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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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 positions corresponding to N31, G179, L267, C332, C335, C351, and C354 of SEQ ID NO: 7.

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

[0387] In some embodiments, the modified TnpB polypeptide comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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 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 polypeptide has the ability to recognize and bind to DNA molecules, but loses the ability to cleave DNA molecules such as double-stranded DNA, ie, is a disarmed TnpB polypeptide.

[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, 340 nucleotides preceding the right end (RE) of the IS from which TnpB is derived. In some embodiments, the backbone is modified by removing a portion or all of one or more stem structures in the backbone. In some embodiments, a portion or all of the first stem structure starting from the 3' end is removed.

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

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

[0392] 6.4. Fusion System

[0393] The present disclosure provides a gene editing system comprising:

[0394] - a fusion polypeptide of the present disclosure, for example, comprising a TnpB polypeptide having RNA-guided endonuclease activity or a functional fragment thereof, or a modified TnpB polypeptide fused to a fusion partner, or a polynucleotide comprising a nucleotide sequence encoding the fusion polypeptide or a functional fragment thereof, and

[0395] - A guide polynucleotide, such as a guide RNA (gRNA), comprising a targeting region capable of hybridizing to a nucleotide sequence of interest and a framework region capable of binding to a TnpB polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding a 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 a microorganism selected from the group consisting of Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas intermedius, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc sp. PCC7120, Clostridium botulinum type C C-Stockholm phage c-st, Methanosarcina acetivora, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Raoultella ornithinolyticus, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Aerobacillus pallidum, and Bacillus thuringiensis.

[0398] In some embodiments, the TnpB polypeptide is encoded by a tnpB gene in an insertion sequence (IS) selected from the group consisting of ISEfa4, ISAs26, ISCpe2, ISMma22, ISBce3, ISAeme8, ISTfu1, ISCco1, ISSoc3, ISTel2, ISNsp3, ISCbt1, ISMac7, ISEc46, ISSen6, ISHah11, 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 consists 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, insertions, deletions, and / or additions. In some embodiments, the variants are conserved at positions corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO: 7.

[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, 350 or more consecutive amino acids 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 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, 350 or more consecutive amino acids 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.

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

[0403] In some embodiments, a TnpB polypeptide of the present disclosure may recognize a TAM of CCAT, CTAC, TGAC, TGAT, TTAC, TTAG, TTAA, TTAT, ACAT, TTTAT, TTTAA, or TTGAT.

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

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

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

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

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

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

[0410] In some embodiments, the TnpB polypeptide or functional fragment thereof disclosed herein is capable of RNA-guided 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, 18, 21, 23 or 24. In some embodiments, the TnpB polypeptide is a functional variant consisting of an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, 18, 21, 23 or 24. The variant may differ from SEQ ID NO:7, 18, 21, 23 or 24 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, insertions, deletions and / or additions. In some embodiments, the variant is conserved at positions corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354 and D361 of SEQ ID NO:7.

[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, 350 or more consecutive amino acids of 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, 350 or more consecutive amino acids of SEQ ID NO: 7, 18, 21, 23 or 24.

[0413] In some embodiments, the TnpB comprises 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 embodiments, the recombinant gene editing system comprises a first polynucleotide and a second polynucleotide, the first polynucleotide comprises a nucleotide sequence encoding a fusion polypeptide, and the second polynucleotide comprises a nucleotide sequence encoding a gRNA.

[0444] In some embodiments, the recombinant gene editing system further comprises a heterologous polynucleotide, such as an expression cassette, a transgene, a donor DNA, or a polynucleotide modification template.

[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, 340 nucleotides preceding the right end (RE) of the IS from which TnpB is derived. In some embodiments, the backbone is modified by removing a portion or all of one or more stem structures in the backbone. In some embodiments, a portion or all of the first stem structure starting from the 3' end is removed.

[0446] In some embodiments, the guide polynucleotide comprises an additional nucleotide sequence at the 5' end of the backbone. In some embodiments, the additional nucleotide sequence can recognize and / or bind to an additional nuclease, such as a TnpB polypeptide or a Cas nuclease of the present disclosure.

[0447] In some embodiments, the modified TnpB polypeptide comprises a modification in a DDE motif compared to a parent TnpB polypeptide, wherein the parent polypeptide comprises an N-terminal HTH domain, a central domain, a C-terminal zinc finger domain, and a DDE motif.

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

[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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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 positions corresponding to N31, G179, L267, C332, C335, C351, and C354 of SEQ ID NO: 7.

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

[0480] In some embodiments, the modified TnpB polypeptide comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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 positions corresponding to G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO: 7.

[0481] In some embodiments, the modified TnpB polypeptide has the ability to recognize and bind to DNA molecules, but loses the ability to cleave DNA molecules such as double-stranded DNA, ie, is a disarmed TnpB polypeptide.

[0482] In some embodiments, the fusion partner is a polypeptide that provides an activity that indirectly increases transcription by acting directly on the target DNA or on a polypeptide associated with the target DNA (e.g., a histone or other DNA binding protein).

[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, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity.

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

[0485] In some embodiments, the fusion partner is a polypeptide that directs single or multiple base editing in a polynucleotide sequence, such as a site-specific deaminase that can change 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 ingenomic DNA without double-stranded DNA cleavage." Nature 533(7603)(2016):420-4).

[0486] In some embodiments, the fusion partner is an active (double-strand break generating), partially active (nicking enzyme) or inactive (unable to cut) TnpB endonuclease and deaminase (such as, but not limited to, cytidine deaminase, adenine deaminase, APOBEC1, APOBEC3A, BE2, BE3, BE4, ABE, etc.). In some embodiments, the fusion partner includes a base editing repair inhibitor and a glycosylase inhibitor (e.g., a uracil glycosylase inhibitor (to prevent uracil removal)).

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

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

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

[0490] 7. Gene Editing Methods

[0491] The present 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 the present disclosure that targets a nucleotide sequence in the polynucleotide.

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

[0493] The present disclosure provides a method for modifying a genomic sequence in a cell, comprising the steps of introducing a disarming system of the present disclosure and a gene editing system targeted to a genomic sequence into the cell, wherein the nucleotide sequence targeted by the disarming system is close to the genomic sequence.

[0494] Methods for introducing polynucleotides or polypeptides or polynucleotide-protein complexes into cells or organisms are known in the art and include, but are 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, mesoporous silica nanoparticle (MSN)-mediated direct protein delivery, 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 genome of AAV (about 4.7 kb) and the large size of Cas polypeptide (more than 1000 amino acids), the delivery of Cas systems using recombinant AAV (rAAV) is limited, and it is generally not possible to deliver a single vector-encoded Cas-fusion system (fusion and gRNA). Compared to the Cas system, due to the much smaller size of the TnpB polypeptide, the gene editing system with the TnpB polypeptide and the TnpB fusion can be delivered in a single rAAV.

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

[0497] In some embodiments, the genome comprises a second expression cassette encoding a guide RNA (gRNA), wherein the guide RNA (gRNA) comprises a targeting 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. In some embodiments, the second expression cassette comprises a promoter and a terminator.

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

[0499] In some embodiments, the genome comprises about 4,500 to about 4,700 nucleotides.

[0500] 8. Method for screening TnpB polypeptides having RNA-guided endonuclease activity

[0501] The present disclosure provides a method for screening the double-stranded DNA cleavage activity of a TnpB polypeptide, comprising the following steps:

[0502] - providing a candidate TnpB polypeptide from a microorganism;

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

[0504] - providing a target DNA comprising a nucleotide sequence that hybridizes with the nucleotide sequence of the targeting region and a TAM recognized by the TnpB polypeptide, wherein the TAM consists of 4 or 5 consecutive nucleotides adjacent to the 5' end of the IS;

[0505] - contacting the TnpB polypeptide with the gRNA and target DNA; and

[0506] - Detection of cleavage on target DNA.

[0507] In some embodiments, the framework 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 framework 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 may be provided as an isolated polypeptide or by expression of a polynucleotide encoding the polypeptide. In some embodiments, the TnpB polypeptide is provided by a first polynucleotide, preferably a DNA, comprising a first nucleotide sequence encoding the TnpB polypeptide.

[0510] The gRNA can be provided as an isolated RNA molecule or can be provided by transcription of a DNA molecule encoding the gRNA. In some embodiments, the gRNA is provided as a second polynucleotide, preferably a DNA, comprising a second nucleotide sequence encoding the gRNA.

[0511] The first and second polynucleotides can 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 operably linked to a first promoter. In some embodiments, the second nucleotide sequence is operably linked to a second promoter.

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

[0513] In some embodiments, contacting the TnpB polypeptide with the gRNA and the target DNA comprises introducing a first and a second plasmid into a host cell comprising the target DNA. Example

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

[0515] This example was conducted to screen for TnpB polypeptides having endonuclease activity.

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

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

[0518] A reporter plasmid (Kan+) carrying oligonucleotides containing the target nucleotide sequence and the relevant TAM, flanked by EcoRI and XhoI restriction sites, was ordered from Tsingke (Beijing, China). Briefly, the oligonucleotides and the pCB457 plasmid were digested with EcoRI and BamHI for 1 h at 37°C. The digestion products were separated and ligated using T4 ligase according to the manufacturer's instructions.

[0519] Take the map of ISTfu1 TnpB plasmid pair as an example, Figure 1 shown.

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

[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 1.8 kV, 25 μF, 200 phm. After electroporation, 900 μl of SOC medium was added, followed by incubation at 37° C. for 1 hour.

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

[0523] The viable colonies were counted and the depletion rate was calculated (depletion rate = the number of colonies in the TnpB & reRNA group in the minimum dilution / the number of colonies in the TnpB alone group in the minimum dilution).

[0524] Blank, K+, and Cm+ plates were used to show the efficiency of electroporation. Generally speaking, the negative control showed similar colony counts on these three plates compared to the test group.

[0525] On the K+Cm+ plate, colonies appeared in the negative control and the test group encoding the TnpB polypeptide having no endonuclease activity, while no colonies appeared in the test group encoding the TnpB polypeptide having endonuclease activity.

[0526] A total of 81 TnpB polypeptides were tested, and the results showed that the TnpB polypeptides of SEQ ID NO: 1-29 exhibited endonuclease activity (see Figure 2 ), cleaving the target site in an RNA-guided manner with different depletion ratios (see Figure 3 ), while the 52 TnpB polypeptides encoded by SEQ ID NOs: 59-110 showed no endonuclease activity (data not shown).

[0527] TnpB polypeptides and gRNAs with RNA-guided endonuclease activity and their TAMs are shown in Table 1 .

[0528] Table 1 TnpB polypeptides with desired activities

[0529]

[0530]

[0531] Example 2. Characterization of TnpB polypeptide

[0532] Sequence analysis

[0533] The amino acid sequences of TnpB polypeptides showing endonuclease activity were aligned (Clustal Omega). The results showed that the DDE motif is conserved in TnpB polypeptides (see Figure 4 and 5 Although the DDE motif in ISBce3 TnpB and ISCbt1 TnpB does not completely match the other motifs, it is actually present and indicated in the amino acid sequence (see Figure 5 ). Notably, TnpB polypeptides with RNA-guided endonuclease activity are generally conserved at positions corresponding to N31, G179, D181, E265, L267, C332, C335, C351, C354, and D361 of SEQ ID NO: 7, except that ISCbt1 (SEQ ID NO: 12) differs from the other polypeptides at positions corresponding to L267, C332, C335, C351, and C354 of SEQ ID NO: 7.

[0534] For clarity, the alignment between the 25 newly identified TnpB polypeptides and the ISDra2 TnpB polypeptide is shown in Figure 12 As shown in A.

[0535] 2.2. DDE motif is essential for endonuclease activity

[0536] Variants of the TnpB polypeptides of SEQ ID NOs: 1-22 were tested as described in Example 1, wherein the first D residue of the DDE motif was replaced by A. The results showed that the variants substantially lost their endonuclease activity (see Figure 2 and 3 ), indicating that the DDE motif is essential for endonuclease activity and that disarmed TnpB polypeptide (dTnpB) can be prepared by introducing mutations in the DDE motif.

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

[0538] Variants of the TnpB polypeptides ISTfu1 (SEQ ID NO: 7), ISDgel0 (SEQ ID NO: 18), ISAba30 (SEQ ID NO: 21) and ISDra2 were tested as described in Example 1, wherein the amino acid corresponding to N31 of SEQ ID NO: 7 was substituted with A. The results showed that the variants substantially lost their endonuclease activity (see Figure 12 B), indicating that conserved amino acid residues such as the amino acid residue corresponding to N31 of SEQ ID NO: 7 are essential for endonuclease activity and that disarmed TnpB polypeptides (dTnpB) can be prepared by introducing mutations in amino acids conserved among TnpB polypeptides.

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

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

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

[0542] Plasmid sets were constructed, each set 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] Briefly, in order to construct a reporter plasmid, oligonucleotides containing the target sequence and the relevant 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 a pUC19 plasmid under the control of a U6 promoter, and the TnpB plasmid was constructed by inserting the coding sequence of the TnpB polypeptide (see Table 1) into pcDNA3.1. The maps of the above three plasmids are shown in Figure 2. Figure 7 shown.

[0544] According to the manufacturer's instructions, 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 Invitrogen 2000 reagent (Invitrogen). The resulting cells were analyzed by flow cytometry (LSRFortessa, BD bioscience).

[0545] like Figure 8 As shown, 5 of the 29 TnpB polypeptides (ISTfu1, ISDgel0, ISAba30, ISAam1, and ISYmu1) exhibited RNA-guided cleavage in eukaryotic cells, i.e., displayed GFP signals in the presence of TnpB polypeptide and gRNA.

[0546] Example 4. TAM preference of ISTful TnpB polypeptide

[0547] The predicted TAM sequence of ISTfull TnpB polypeptide is 5'-TGAT, which is similar to the TAM sequence of ISDra2 TnpB (5'-TTGAT). 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 plasmid encoding ISTful TnpB polypeptide or ISDra2 TnpB polypeptide and their respective guide RNAs were co-transfected with the reporter plasmid into 293T cells, and the resulting cells were detected by flow cytometry.

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

[0550] like Figure 9 As shown, the ISTfu1 TnpB peptide recognized all four TAMs and showed high cleavage efficiency for TAM TTGAT and CTGAT, while the ISDra2 TnpB peptide only recognized TTGAT. In addition, the ISTfu1 TnpB peptide exhibited a 2-fold higher cleavage efficiency than the ISDra2 TnpB peptide.

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

[0552] This example was performed to verify that the TnpB polypeptide of the present invention directed the cleavage of endogenous genes in human cells by RNA. The gRNA backbones used in this example were those listed in Table 1.

[0553] 5.1. Editing of ISTfu1 TnpB in hDNMT1

[0554] As described in Example 3, a plasmid encoding the ISTful TnpB polypeptide (SEQ ID NO: 7) and a gRNA containing the targeting region of SEQ ID NO: 142 (target sequence 1 in hDNMT1) 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 harvested and genomic DNA was isolated using an isolation kit (DP201, Bioteke Corporation, Beijing) according to the manufacturer's instructions. Genomic DNA was tested by Surveyor assay to determine cleavage efficiency, and genomic DNA from untreated 293T cells was used as a control.

[0555] The Surveyor assay was performed with reference to Guschin et al. (2010) (Guschin et al., A-Rapid and General Assay for Monitoring Endogenous Gene Modification, Methods in Molecular Biology, 2010, 649:247-256). Briefly, 100 ng of genomic DNA was used in a 25 μL PCR reaction using AccuPrime Taq polymerase (Invitrogen, USA) and primers of SEQ ID NOs: 218 and 219. PCR conditions were as follows: 94°C for 2 min; 30X (94°C for 20 s, 60°C for 20 s, 68°C for 40 s); 68°C for 3 min; hold at 4°C.

[0556] 6.5 μL of PCR product was then denatured, annealed with 3 μL of 1X AccuPrime Buffer II, and then digested with 0.5 mL Surveyor nuclease (Integrated DNA Technologies, IDT, USA). The samples were run on a 10% acrylamide TBE gel, stained with ethidium bromide for 10 minutes, rinsed with water, and then exposed to a Bio-rad gel imager. Band intensities were quantified using Image J 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 A, lanes #1, #2, and #3 showed cleavage by Surveyor nuclease (indels% was 14.5%, 16.6%, or 20.0%), indicating that the ISTful TnpB polypeptide can achieve RNA-guided cleavage of human DNMT1 in human cells.

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

[0560] As described in Example 3, plasmids encoding ISDgel0 TnpB polypeptide (SEQ ID NO: 17) and a gRNA containing a targeting region of SEQ ID NO: 143 (target sequence 1 in hTET1), 144 (target sequence 1 in hTET2), or 145 (target sequence in hHPRT) and a backbone of SEQ ID NO: 127 were constructed and transfected into 293T cells. A plasmid encoding spCas9 and a gRNA containing the same targeting region was used as a reference.

[0561] Transfected cells were tested by Surveyor assay of genomic DNA as described in Example 5.1 using the primers listed below.

[0562] target Forward primer Reverse primer 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 Figure B, the ISDgel0 TnpB polypeptide achieved RNA-guided cleavage of hTET1, hTET, and hHPRT, thereby introducing indels into them, indicating that the ISDgel0 TnpB polypeptide can achieve RNA-guided cleavage of hTET1, hTET2, and hHPRT in human cells.

[0564] ISAba30 TnpB Editing in hDNMT1

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

[0566] Transfected cells were tested by Surveyor assay of genomic DNA using primers of SEQ ID NOs: 226 and 227 as described in Example 5.1.

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

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

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

[0570] Transfected cells were tested by Surveyor assay of genomic DNA as described in Example 5.1 using the primers listed below.

[0571] target Forward primer Reverse primer 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 polypeptide achieved RNA-guided cleavage of hTET1 and hTET2, thereby introducing indels into them, indicating that the ISAam1 TnpB polypeptide can achieve RNA-guided cleavage of hTET1 and hTET2 in human cells, and the indels% is comparable to or even higher than that of spCas9.

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

[0574] As described in Example 3, plasmids encoding the ISYmul TnpB polypeptide (SEQ ID NO: 24) and a gRNA comprising a 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 were constructed and transfected into 293T cells.

[0575] Transfected cells were tested by Surveyor assay of genomic DNA as described in Example 5.1 using the primers listed below.

[0576] target Forward primer Reverse primer 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 polypeptide achieved RNA-guided cleavage of hDNMT1, hDNMT3b, hTET, and hPGK1, thereby introducing indels into them, indicating that the ISAba30 TnpB polypeptide can achieve RNA-guided cleavage of hDNMT1, hDNMT3b, hTET, and hPGK1 in human cells.

[0578] Comparison of ISAam1, ISYmu1, and ISDra2 in Human Gene Editing

[0579] Plasmids encoding ISAam1 TnpB polypeptide (SEQ ID NO: 23) / ISYmu1 TnpB polypeptide (SEQ ID NO: 24) / ISDra2 TnpB polypeptide and gRNA containing the targeting region in 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] Transfected cells were tested by Surveyor assay of genomic DNA as described in Example 5.1 using the primers listed below.

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

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

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

[0586] Table 3. gRNA backbone of ISDra2

[0587] gRNA The sequence of the backbone (SEQ ID NO: g97 153 g115 154 g129 155 g149 156 g174 157 g199 158 g231 159 g248 160 g270 161 g301 162

[0588] Table 4. gRNA backbone of ISTfu1

[0589] gRNA The sequence of the backbone (SEQ ID NO: 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. gRNA backbone of ISAba30

[0594] gRNA The sequence of the backbone (SEQ ID NO: 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. gRNA backbone of ISAam1

[0596] gRNA The sequence of the backbone (SEQ ID NO: 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, ISDgelO, ISAba30, ISAam1, and ISYmu1 TnpB polypeptides was performed as described in Example 3. Figure 11 shown.

[0601] Specifically, ISDra2 TnpB is able to cleave target sequences with a 129nt gRNA backbone ( Figure 11 A); ISTfu1 TnpB polypeptide can cleave the target sequence with a 139nt gRNA backbone ( Figure 11 B); ISDge10 TnpB polypeptide can cleave the target sequence with a 122nt gRNA backbone ( Figure 11 C); ISAba30 TnpB polypeptide can cleave the target sequence with a 101nt gRNA backbone ( Figure 11 D); ISAam1 TnpB polypeptide can cleave the target sequence with a 100nt gRNA backbone ( Figure 11 E); ISYmu1 TnpB polypeptide can cleave the target sequence with a 120nt gRNA backbone ( Figure 11 F). TnpB polypeptide can also cut the target sequence with a longer gRNA backbone ( Figure 11 A-11F), demonstrating that RNPs can be formed using longer gRNA backbones.

[0602] ISDra2, ISTfu1 and ISDgel0 TnpB polypeptides were subjected to backbone-based sequencing (based on the backbones of SEQ ID NOs: 157, 166 and 178, respectively, see Figure 10 A-10C, right) with a modified frame design at the right end (see Figure 10 A-10C, right panel), and RNA-guided cleavage was performed as described in Example 3. Figure 10As shown in A-10C, substitution of the right-terminal nucleotides inhibits RNA-guided cleavage, substitution of two right-terminal nucleotides greatly inhibits RNA-guided cleavage, and addition or deletion of one or more nucleotides at the right end greatly inhibits or even eliminates RNA-guided cleavage, indicating that the right end of the gRNA backbone is crucial for RNA-guided cleavage of the TnpB polypeptide.

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

[0604] This example was performed to demonstrate that the editing efficiency of TnpB polypeptide is superior to that of small Cas nuclease.

[0605] Specifically, the in vitro and in vivo activity and specificity of the ISAam1 and ISYmu1 TnpB polypeptides were tested and compared with five well-established CRISPR-Cas editors, including two optimized Un1Cas12f1 variants (termed Un1Cas12f1 and CasMINI), AsCas12f1, optimized Nme2Cas9 (Nme2-C.NR), and SaCas9. To make these seven systems comparable despite their different TAM / PAM requirements, we carefully selected genomic regions where the gRNAs for each system could be designed to target sequences that overlapped 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, which 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 polypeptides is significantly higher than that of the mature small Cas editors (Un1Cas12f1, CasMINI, AsCas12f1 and Nme2-C.NR) and is comparable to that of SaCas9.

[0608] To further reduce the confounding effect of inter-target distance, we performed a comparison in the HEK293T cell line using overlapping (>15 bp) gRNAs targeting three endogenous targets. The previous pattern was largely replicated, with ISAam1 and ISYmu1 TnpB exhibiting higher activity than the Cas12f nuclease together with SaCas9.

[0609] rAAV encoding the genome editing system was prepared as previously described (see Ran, FA et al. 2015, In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186-191). Briefly, a nuclease expression cassette driven by a CMV promoter and a gRNA / reRNA expression cassette driven by a human U6 promoter were cloned between the ITRs (see Figure 16 A) and used to prepare recombinant AAV2 and AAV8. HEK293T cells were plated in 150mm culture dishes 12 hours before transfection; 30μg of helper plasmid, 15μg of AAV2 plasmid and 15μg of expression plasmid were transfected using polyethyleneimine, and 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 5×10 4 gc / well were seeded in 48-well plates. AAV2 was added at 10 4 The cells were added to the cells at a multiplicity of infection of 1000 gc / well. Cells were harvested 4 days after transduction for genomic DNA extraction and editing efficiency analysis by next generation sequencing. Targeting the Rosa26 locus, the ISAam1 TnpB and Cas9 systems showed significant activity ( Figure 16 A) Given that AsCas12f has the lowest average editing activity among Cas12fs and Nme2-C.NR generally has lower activity than saCas9, we excluded it from in vivo experiments to avoid unnecessary sacrifice of more mice.

[0611] We then delivered a single AAV8 vector encoding each of the five different editing systems into mice and analyzed the editing activity in the target organ, the liver. All experiments related to animal work described in this study were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Welfare and Research Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences. The mouse strain C57BL / 6J was obtained from Vitalriver. Six-week-old female C57BL / 6J mice were injected via the tail vein with 5x 10 11 gc. 14 days later, the mice were sacrificed and liver tissue was collected for genome extraction. This in vivo result roughly reproduced the above in vitro data, in which the TnpB and SaCas9 systems showed relatively high activity ( Figure 16 B).

[0612] To quantify the level of editing specificity or off-target, we used a candidate-based assay in mouse N2a cells and an unbiased genome-wide assay by iGUIDE-seq in human HEK293T cells (see Nobles, C.L. et al., 2019, iGUIDE: an improved pipeline for analyzing CRISPR cleavage specificity. Genome Biol. 20, 14). Briefly, 0.5 × 10 cells were transfected with 1 μg of nuclease plasmid, 500 ng of gRNA plasmid, and 50 pmol of double-stranded oligodeoxynucleotide (dsODN) using the Lonza 4D system (program CM-130). 6 HEK293T cells. Cells were collected 3 days after nucleofection for genomic extraction. Genomic libraries were prepared and sequenced. Specifically, for the Rosa26 and Angptl3 loci in mice, we predicted 7 potential off-target sites for the system, quantified the indel frequencies of the top 2 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 the specificity. Among the three loci, ISAam1 had the lowest off-target ratio for both loci ( Figure 17 A and 17B), while ISYmu1 also exhibited low levels of off-target editing (no off-target editing at one locus and the second or third lowest off-target editing levels at the remaining two loci).

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

[0614] sequence

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

[0616] MEQLKAYKFRIYPTEEQEIFFAKSFGCVRKVYNLMLDDRKKAYEEVKNDSSKKMTFPTPAKYKKEFPFLKEIDSLALANAQLNLDKAYKNFFRDKSVGFPRFKSKKNPVQSYTTNNQNGTVALIDSKFIKVPKLKSLVRIKLHRQPKGIIKSATISRHSSGKYYISLLCKEEIIELPKTNSTIGIDLGIMDFAILSDGQKIDNNKFTSKMEKKLKREQRKLSRRALLAKNKGTNLFEAKNYQKQKRKVARLHEKVMNQRTDFLNKLSTEMIKNHDIICIEDLNTKGMLRNHKLAKSISDVSWSKFVTKLQYKADWYGRKIIKVDKWFPSSQICSECGHKDGKKSLEIREWTCPVCHTHHDRDINASINILTEGLRLHSMGLA

[0617] SEQ ID NO:2 is the amino acid sequence of As26 TnpB

[0618] MQILKAYKFRLEPTEQQAQRLRQLGGCARYVWNHGLAETKRILDAGEKLPSAFELNRMLTSWKKDPEMAFLAEAYTDNLQQKLKDLHGAWMRCFDKKLAAKAPVFKKKSDNRDAIRFVNFDKYCRLEKGRVKLPAGLAWVKFRQSQPVEGKIKNATVSQAAGHWYISFQVEQETLEPIHPSGSMVGLDAGIAKLATLSDGTVFEPVNSFKTNQTKLARLQRQLSKKVKFSSNWKKQKAKIQRLHSHIANTRRDYLHKVTTTISKNHAMIVIEDLKVASMSKSAAGTTSQPGRNVRAKSGLNRSILDQGWFELRRQLEYKQAWRGGQVLAINPAYTSQQCACCGHTAKDNRQTQSQFKCAACGYEANADVNGARNILAAGHAVLACGGVVQSGRPMKQEPGTARAAITQ

[0619] SEQ ID NO:3 is the amino acid sequence of Cpe2 TnpB

[0620] MIRKKAYKFRIYPTKKQAELINKTIGCCRFVFNYSLGVQKTKDNYWNIVEEMVQQGYFQENNWKGEFFNKANSIKDIAKLKKSYDWLKEVDSIALQASVENLDAGYDKYYKKTGSRPKFKSKKNEIQSYTTKLVKAKGNVNIEIVGKGIKLPKLGLVKIENSRNVDGNIKRVTVSRTQSGKYFASILCDVNIQELPKIDKKVGVDVGLKTFAVCSDGYEEANPKHFRKAEKRLIKLQRDLARKEYNSKNYHKNRIMIAKLHERIANQRMDFLQKFSTKLIRENQSIAIEDLRVSNMLKNHKLAKVISEASWSEFRRMLEYKAEWYGREIVIAPPDYASSQLCSECGYKNIEVKNLGLREWVCPECGTHHQRDLNASINLEKLIA

[0621] SEQ ID NO:4 is the amino acid sequence of Mma22 TnpB

[0622] MLKVLRYRVYPTKAQKTTMNNTLELCRWTYNETLALRKDAWESEQKNIGYYDSKKMIPEWKKEKPELKTVHSQVLQEVIKRVDLAFQAFFRRVKTGEKAGYPRFRGYGRYDSFTYTQSGFSLDSDRLTLSKIGDIKIKLHRPVEGEIKRLNVRKMPTGKWFVSFLVETDTPLELQKTGLSVGVDVGIKSFLTLSDGNYVPNPRFFVTEEKFLAKVQRKLSKLEKGTPERAKALKAVQRVHERISNKREDFVQKVSLNLVRNYDLITFEDLNIKGMIKNHKLAKHIADVAWNKLVTITSYKAEWAGKRVELVNPCNTSQMCSGCGEIVKKELSERIHSCPYCGLTLDRDHNAAINIMRLGLQSLQNSGRCPSLQ

[0623] SEQ ID NO:5 is the amino acid sequence of Bce3 TnpB

[0624] MSQTITVKVKLLPTKEQIQLLEQSSYEYIKVINTLVSEMAEAKKSTKKSTKDVEANLPSAVKNQAIKDAKSVFSTKVKKSKYKVIPILKRPVCVWNNQNYSFDFTHISVPLMVNGKSTRLKVRALLTDKYNRNFNLLTHKLGTLRITKKSNKWIAQISVTIPTNEKTGTKILGVDLGLKVPAVAITDDEKVRFFGNGRQNKYMKRKFRSVRKKLGENKKLNAIRQLDDKEQRWMQDQDHKVSREIIDFATDNNISVIRLEQLTNIRQTARTSRKNEKNLHTWSFYRLAQFIEYKANLVGIKVEYVNPAYTSQTCPKCSEKNKAPDRKYKCRCGFEKHRDIVGAMNIRYATVIDGNSQSA

[0625] SEQ ID NO:6 is the Aeme8 TnpB amino acid sequence

[0626] MTLRRATKVRIYPTDEQAAFLNAQFGAVRFAYNKALHIQRHMFKRHGISLKPKRDLKPMLAVAKKSRKYGWLKEYDSLALQQAVINLDKAFANFFNPKLKARMPTFKSKRGRQSSYHPNGKVLTAAIQLPKMTPIRAVIHRDIIGVVSSITVSRGPTGKYYASILCEDGCEAPAKPTFITDVTGCDMGLSHYLIESSGKKIANPRHLINASRNLRRKQKALSRKKKGSANRGKARRQLAALHERVANARADFQHKLSHTMVDENQAVIVETLKSANMMKNHKLARAIGDAGWHGFIMKLEYKAQAAGRHLIKLDQWFASSKLCSDCGHKMPDMPLHQRQWVCPACGAGHDRDINAAMNIQQQGILELKAAGHVVSTHGGQR

[0627] SEQ ID NO:7 is the Tfu1 TnpB amino acid sequence

[0628] MLTGRRYLLAFTPEQEEFAEKIGDACRVVWNTALEQRRQYRRRGAFIGYVEQARQMAEAKKDFPWLAEAPSHTLQQTLRDLERACKTHGTFKVRWRSKRKNAPTFRFPDPKQITVERISRRWGRVKLPKLGWTRFRWTRPLGGQLRNATVLKDGGRWYISFCVEDGLMESAPNGKPPVGVDRGVTVAVATSSGWMRDREFVTPGEAVRLKRLQQQLARQRKGSNRRAATRAKLTKLNARIRARRTDFLAWTANRLTCDHGLVVVEDLNIRNMTASAKGTLAEPGRNVAAKAGLNRAILAKGWGGLLTALEHKARYHGSRILRVPPAFTSQTCHACGHCAPDNRESQAEFRCRACGHQANADVNAAKNILAAGLAVTGRGDLAVGRSVKRQPPKVEAA

[0629] SEQ ID NO:8 is the Cco1 TnpB amino acid sequence

[0630] MQITKGFKYRLYPNKEQSLKLSHQCFIYNQAYNICLNLQQEQWLKNKELDKKERTYLSATQIDNQVKLALKARNLEFKSVVAQQARINANRALEQAIKDKNKGFPKFKNSKLAKQSFNWNNQGYQILEFNDKFKIFRLMSMPIKMRYHRALPNNYKLNQITISKTHNKYFVSFSITYDEPSVVLMSSNKIDINRAVGIDMNINEIALSNGALLLTCSKSINKAKYSTTFKRLMRKQSKRVLKAKKDKIKLGSNFRKTQIRLNKIYERASNIKKDLAHKISNQIVKNFDLIVVENLKVKNMTKRAKLKNVKAKSGLNKSILDTSFYQFLSFVEYKAMHNGKVFTKIEPQYTSKACCKCGTIKENLTLKDRVFTCDNCGYVEHRDINASKNILDRGLKSFGLGTSLLDYKQEALRVS

[0631] SEQ ID NO:9 is the Soc3 TnpB amino acid sequence

[0632] MIITHEYRILPSDDQAALMTEWLELLRRQWNDALGQRLDWLTATRCPIDRCSLVSCPLPVSEPPLEPNYYRQAGSLKQIKQLFPAYRGIYAEVLQQNLMRLDKAWKAWREPDSTGKRRGRPRFKKAGELRSFTFPRINCPKAGAHLEGETLRLSKIGSMPVVLHRPLPEGFVPKTCTVVRKADGWYVCITLEDKSVPSPEPVPIKKAVGIDVGLDRFLTTSDGEVVPIPRHYRRAQKHLARQQRQLSRKVKGSANWKRQATKVACLQLHVARQRKAFHYQVAHWLVEQYDLLVVEDLNVRGLARTRLAKSILDAAWGRFLDILTAVAVKRGKQVLRVDPRGTSQNCCVCEERVPKTLSERVHDCPRCGSWDRDLNAAIEILKRGLRAVGLPLSGCGGSWFTSPLKQQLREVILGSSRLQPVRA

[0633] SEQ ID NO:10 is the Tel2 TnpB amino acid sequence

[0634] MKARYRYRFYPTDQQRQRLAQLFGCVRVVWNDALAICKQSDSLPKTSELQKLVITQGKKTPERQWLSDVSNVPLQQSVADLGVAYKNFFDSIKGRRKGKKINPPRFKKKTERQSARFTTYGFSIKGEEVYLAKIGNVRPIWSRKLPSKPSSVTVIKDAANRYFLSFVVEIDPVHQPASNPSIGIDLGIKAFATLSTGEKIDGPDYSKLDKKIRRKQRKLARQVKGSQRREKTRLQIAKLHSRISDIRRDFLHKVSTRVVIENQVITLEDLNVSGMVKNRKLARAISLQGWREFRVLVEAKCNKLNREFIVIDRWEPTSQTCSKCGFKWGKVDLSVRSVVCLNCGTEHDRDENAARNIEKVGIGHCHDYKWTQRGSKTTSVASPSEASRIIAL

[0635] SEQ ID NO:11 is the Nsp3 TnpB amino acid sequence

[0636] MKTLKFKLYQHKRNRFLKRMINASGVIYNHCIALHKRYYRMWGKHLSCAKLQAHIAKLRKRNPFWQTVGSQAVQDICQRIEKAYQLFFKHNKKGVRPPGFKKVKKYKSFTLKQAGYKFLGGNRVKIGNRVYQFWKSRQIEGTVKTLTIKRTPLGELFVVVVVDDGIKPEIEIKTGKIAGFDFGLKTFLTCSDGTKIDSPQFFKQSLNAITKASRQHSKKLKGSANRKRARLNLVRKYEDISNRRRDWFWKLAYELTDKFDVLCFETLNLKGMQRRWGRKISDLAFGEFLEILEWVAKKKNKLIVFIDQWYPSTKTCSHCGHVLESIDLSVRSWSCPSCQSVNERDENASKVICAVGASTVGLGDVRLATPAIAV

[0637] SEQ ID NO:12 is the Cbt1 TnpB amino acid sequence

[0638] MAKSKTSSYILTLKLKTEKYQEDILNKRLEISRNIYNSCLGEILKRYKHTRELKDYRKEYKNIIKMSKDKERSKKFNELNKKYDLTEYSLHTYVKPIQKHFKDNVDSFTAQKIATRVFNSFQKLMFHQAKRVYFKRYGELNSVEGKSNKTGIRFQDNSLIWNGLKIPVIIKTNDIYVQMSLENRVKYCRIIRRIVKGKIKFYVQLILEGIPPMKINKTTGEIKNKIGKGNVGIDIGTRTIAVSSENDVKLLELAPEIDNIENQKRILNRKLDRQRRANNPNKYNEDGTINRNNKDKWIKSNRYIKTQNKLKEIQRKQAEIRKQSHYRLINRLLLLGNRFLVETMNYKGLQARVKETTINEKTGRFNKKKRFGKSLANKAPSMFLTMLDNKLKWNNTQLFKIDTKKCKASQYNHFTNEYCKKELKDRWNEDIQIQRDMYSAFLIMNVVGKTLDKIDRDLCIETYDNFRRLHDKEIERLKELKKNGYKLISSMGI

[0639] SEQ ID NO:13 is the Mac7 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 is the amino acid sequence of Hahl1 TnpB

[0646] MIQRKATYRLYPTAEQLAALEHQLWVHCLLWNEALAERRWAWSERQESLGFSAQCKRLTEWRSQSKLLSGLNAQSEQVTLKRLDLAFQHFFRRVKQGETPGFPRFKSLHRFKGWGYKTHGDGWRLLTNEQMQHGTLRLSGVGAVAVRGRPRTPGTPKTCEIIKRGDRWYASITLNCEPVRECGEKMGGLDWGLETFATVATAQGAETIDNPRHLAGTLDAIRGVQREISRKEEAAKKASGRSRGFPISNRLRKAYDELRRLHRKVANQRHDFLHQVSARLIKEFSALGLEALSIRNMTAKGGQHKRGLNRGILDAAGGAFHQLLGYKAEEAGAWAVEAPTRQIKPSQTCHACGQQEKKPLGQRWHSCPCGASCSRDENAARVLLAWLERSLSGREPADAWREVRPGHPLDESALPSKRETHAIA

[0647] SEQ ID NO:17 is the amino acid sequence of Kpn-69 TnpB

[0648] MLIQKAYKYRLNPTDAQAQRLRQMCGCARFVWNYALAETQRILEAGQKLPSAFELNKMLTGWKKLPDLAFLSDAYTDNLQQKLKDLHGAWIRCFDKKLAAKAPVFKRKTHGNDSVRFVNFDKYCQLEYRRVKLPSGLGWVTFRQSRAVVGKIKNATVSQHAGGWYISFQVEMEVEQPAHPSTSAVGLDAGICKLVTLSDGTVFEPVNSFKQNQSRLARLQRQLARKVKFSANWKKQKARISRLHSHIANIRRDYLHKVTTTISKNHAMIVIEDLKVSNMSKSAAGTVDEPGRNVAAKSGLNRAILDQGWYEMRRQLEYKQRWRGGEVLAVNPAYTSQKCARCGHTVKENRQSQAVFVCVACGYEANADINGAQNILAAGHAVLSGINPGRARKAA

[0649] SEQ ID NO:18 IS the TnpB amino acid sequence of Dge10

[0650] MTTHRKVYRYRIEPTPVQESKLYMLAGSRRFVFNWALARRREHYAETGKTLGYNAQAGELTALKNQEETSWLKESDSQLLQQALKDVERAFVNFFEKRARFPRFKSKKTDTPRFRIPQRVRIEGSRVYVPKVGWVKLRKSQEIEGKTKSATFKREADGHWYVLLVSEFEMPDVPLPPVPESEVVGIDLGLKDFYVLSDGGRKEAPRFARKGQRKLRRAARRHSKCTRGSNRKAKAKRKLARVHRQIANQRKDFVHKATSGLVQQYQGFCIENLSIKGMAKTKLSKSVLDAALGEFRRQLAYKAQWHRKWLAVIDRWFPSSKLCGECGSINADLTLSDREWTCECGAVHDRDLNAARNIKREGLSQIVVAGHAETLNARGEGVRPAIAGSPR

[0651] SEQ ID NO:19 IS the TnpB amino acid sequence of Kpn85

[0652] MLIQKAYKFRLNPTDVQAQRLRQLCGCARFVWNYGLAETQRILETGQKLPSAFELNKMLTGWKKLPDLAFLSDAYTDNLQQKLKDLHGAWKRCFDKKLAAKAPVFKRKSHGNDSVRFVNFDKYCQLEYRRVKLPSGLGWVTFRQSRDIVGKIKNATVSQHAGSWYISFQVEMEVDQPAHPSTSAVGLDAGVSKLATLSDGTVFEPVNSFKQNQSRLARLQRQLARKVKFSANWKKQKARISRLHSHIANIRLDYLHKVTTTVSKNHAMIVIEDLKVSSMSKSAAGAADMPGRNVAAKSGLNKAILDQGWYELRRQLEYKQSWLGGQVLAVNPAYTSQKCACCGHRSKENRKSQANFVCLECGYKENADINGARNILAAGHAVLACEVNGASMPSAAGTKRLSNQLATQ

[0653] SEQ ID NO:20 is the Nsp2 TnpB amino acid sequence

[0654] MKTLKFKLYQHKRNRFLKRMINAGGAIYNHCIALHKRYYRMWGKHLNCAKLQAHIAKLRKRNPFWQTVGSQAVQDICQRIEKAYQLFFKHNKKGVRPPGFKKVKKYKSFTLKQAGYKFLDGNRVKIGSKIYQFWKSREIEGTVKTLTIKRTPLGELFMVVVVDEGNKPETKFMTGKIAGFDFGLKTFLTCSDGTTIESPQFFKQSLNAIKKASRQHSKKLKGSANREQARKNLVRRYEDIFNRRRDWFWKLAHELTDKFDVLCFETLNLKGMQRLWGRKISDLAFGELLQILEWVAKNKNKLVVFIDPWYPSSKTCSSCGHILENLDLSIRRWRCPSCQSENDRDGNASLNIKRVGSSTLGVGDVRQSQTAISV

[0655] SEQ ID NO:21 is the Aba30 TnpB amino acid sequence

[0656] MKRLQAFKYEMVPNGQQERQMRRFAGSCRFVYNKALALQKERYERGEKKLGYAGLCKLLTEWRNSSEMAWLADAPVHPLQQTLKDLERAYSNFFAKRADFPRFKKKGCHSSFRYPDPKQIKLEQHNSRIFLPKLGWIRYRKSRDVLGAVKNVTVSLSGGKWFVSIQTEREVEQPIPQGGAVGIDMGIARFATISDGKFYAPLNSFKRHEKRLRCAQQALSRKVKFSNNWQKAKARIQRIHSRIGNARRDFLHKTSTAISQNHAMVCIEDLQVRNMSKSAAGTPEQPGRNVRAKSGLNKSILDQGWFEFRRQLDYKLAWNGGWLVAVPPKNTSRTCPCCAHVSADNRQTQERFECIECGFEGNADVVGAINVLRAGHARLACEVSDAVGSPAAGTHRSDSDQAHCRV

[0657] SEQ ID NO:22 IS the amino acid sequence of Ror9 TnpB

[0658] MHRATKVRIYPTQEQTELLEKQFGAVRFAYNKALYIRSHWYKKHGVSLNNAKHIQPLLPIAKKSRRYSWLKAADSNALQVSLQNLDRAFQNFFEKRAKYPKFKRKHGPQSSYHCSSLKITADTIKIPKISPIRANIHREIVGKVKSITISRAPTGKYYASILADDGQEAPAKPTYITQLTGYDIGLKDYLIRDDGKKTPNPRHLVNAYRRLRRDSKALSRKQKGSANRRKARIRLAAQHDRVANARNDFQHKLSRNIIVENQAVIVETLKTANMMKNKRLARHIGDAAWSGFVAKLAYKAEASGVHLVKLDQWFASSKTCHCCGHKMAEMPLKVREWQCPVCLTHHNRDINAAMNIRLKGITELMAAGHVVKAHGGLRKSVITTVAAYEVGSPAL

[0659] SEQ ID NO:23 IS the amino acid sequence of Aam1

[0660] MVNKSYKFRLYPTKEQEQLLAKTFGCVRFVYNKMLEERIQIYEKFKDDKEALKKQTFPTPAKYKKEFPWLKEVDSLALANAQLNLQKAFQNFFSGRAGFPKFKNRKAKQSYTTNVVNGNIQLSDGYIKLPKLKWVKFKQHREIPAHHIIKACTIKKTKTGKYYVSILTEYEHQPVPKEIQTVVGLDFSMNGLFVDSEGKRANYPRFYRQALEKLAKEQRILSRRKKGSNRWHKQRLKVAKLHEKIANQRKDFLHKKSYELAKQYDCVVIENLNMKGMSQVLNFGKSVHDNGWGMFTTFLQYKLEEQGKKLIKIDKWFPSSKTCSCCDQVKESLSLSERTFRCECGFESDRDVNAAINIKHEGMKRLAIA

[0661] SEQ ID NO:24 IS the amino acid sequence of Ymu1

[0662] LLQHKAYEYRIYPDKKQETLIAKTIGSSRYVYNHFLELWNKEYEETGKGLTYYACSKLLTKLKRDPETVWLCEVDKFSLQNSLRNLSDAFSRFFKGQNEHPQFKSKKSPRQSYTTQYTNNNIAVSGNCLKLPKLGLVKFADSREMKGRILNATVRRKSSGKFFVSILCEEEICELPKTDSSVGIDLGIIDFAVMSDGSRHDNNHFTRQMEERLRREQRKLARRALAAEKRGISLSEARNYQKQRRKVARLYEKVANQRKEYLNKLSTEIVKNHDIICIEDLNVKGMMRNHKLAKSISDVSWTSLVSKLQYKASWYGKEVIRISRWFPSSQICSECGHKDRKKPLHVREWTCPVCHAHHDRDVNAARNILAEGLRIRALTPGS

[0663] SEQ ID NO:25 IS the amino acid sequence of Cytsp1

[0664] MTKQNKAYKFRLYPTDEQAFIIRKTFGCVRFVYNKMLAERIETYESLKEDKEALKKIKHPTPAKYKKEYEWLKEVDSLALANAQLNLDKAYKAFFKGNVKFPTFKRKRHKQSYTTNVVNGNIQLLDGHIKLPKLKLVKIKQHRKIPSEYKIKSCTLSMTASGKYYISILAEYEVEIERKEVKKVVGLDFAMDGLFVDSEGEKANYPKFYRQKLEKLAKEQRKLSRKKKGSSNWNKQRIRVANIQEKVANQRKNFLHHQSKKLVANYDAVVIEDLDMKGMSQALKFGKSVADNGWGMFTSFLQYKLKEQGKQLIKIDKWFPSTQTCSHCGNTQLMPMNIRIYVCSCGLNLDRDYNSALNIKKEGIRLLASA

[0665] SEQ ID NO:26 is the Cvi1 amino acid sequence

[0666] MNKGVKFRAYPNKEQQNLINRTLGCCRLIYNKGLAMRNDAYSNGEKIGYNQTSAMLTNLKKQADFAFLKDVDSIALQQSLRDLDRGFKNFFEKRARHPQFKSKHNNHQSYRTINQGDNIRIIGKYIKLPKLGYVKIKQSMEVGHINNVTVERTPTGKYFVVLNVDFEPEFRPNAGCMIGIDVGIKEFYSDSNGNVVNNPKYLEKSMRKLAREQRRLSRKQKGSNNRNRQRVKVAKVHEKITNQRNDFLQKQSTILVSENQTICIEDLNVKGMIRNHKLAQHIASCSWSKFFAMLEYKAIWYGNDIIKVPTMYPSSQTCSCCGYKNPFVKNLSIREWECPNCHAKHDRDTNASITILNKGLSIA

[0667] SEQ ID NO:27 is the Cvi2 amino acid sequence

[0668] MNKAIKYRVHPTTEQSVMFAKTFGCCRKVYNLMLSDKIDGYKATGKFPTVTPAKYKNDYPYLREVDSLALANKQMDLQEAFRNTFSKSRKKKNGFPKFKSAKHSRKSYTTNNQHGTVAIIDNKYIKLPKIGKVKAVIHRIPDSDWIVKSATVSQESDGKYYISVLFEFDNAINLYVADKTNAIGLDYTSNGLYVDSNGNVGTNHKYYRESHDKLAKAQRRLSRMQGSKKHEIKSNNYIKQLRKVNKIHRYIANQRLDNLHKISTKIANSYDVVCVESLNMRSMANHGFGNGKATLDNGYGMFLSMLEYKLSDRNKYLIKVDKWFPSSQICHCCGKIHPEMKNLTIRTMKCDCGLTMNRDQNAAINILREGLRLLTKVA

[0669] SEQ ID NO:28 is the Aepa1 amino acid sequence

[0670] MAKQNKAFKFRLYPNKEQKIQLAKTFGCVRFVYNKMLEERKETYEKFKNDKETLKQQAFPTPAKYKDEFPFLKEVDSLALANAQINLQNAYKNFFEGRAEFPKFKSKKNKQSYTTNMVNGNIKLENGHIKLPKIKKPMKMNRHREIPADYKIKSCTISKTKTGKYYISILTEYEKDIQSVKIQKVVGLDFAMDGLYVESEQGEKANYPRYYRQALDKLAKAQRILSRRKKGSARWEKQRLKVAKLQEKIANQRKNFLHHKSKELASTYDAVIIEDLDMKGMSQALNFGKSVHDNGWGMFTTFLEYKLKEQGKQLVKIDKWFPSTKKCSCCGKEKEMPLSKRVYECSCGFVLDRDHNSAINIKNEGLRILA

[0671] SEQ ID NO:29 is the Bth16 amino acid sequence

[0672] MTKQNKAYKFRLYPTKDQAHLMRKTFGCVRFVYNRMLAERKEAYEKHKDDKDQLKKQKLPTPAKYKAEFEWLKEVDSLALANAQLNLQTAYKNFFRGQNDFPTFKSKKDRKSYTTNVVNGNIILLNGHIKLPKLKMVRIKQHREIPQGHIIKSCTISMTPTGKYYVSILTEYEKEIVQKEVETVVGLDFAMDQLYVSSEDERANYPKFYREMLDRLAKAQRVLSRRTKGSGRWNKQRIRVAKLHEKVANQRKNFLHHKSKELATHFDVVAIEDLNMKGMSQALHFGKSVADNGWGMFTSFLAYKLHEQGKQLVKIDKWFPSTKTCSSCGNMKNMSLSERVYSCICGVNLDRDYNAAINIKNEAIRLLALV

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

[0674]

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

[0676]

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

[0678]

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

[0680]

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

[0682]

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

[0684]

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

[0686]

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

[0688]

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

[0690]

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

[0692]

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

[0694]

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

[0696]

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

[0698]

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

[0700]

[0701] SEQ ID NO:44 ISSen6 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:48 ISKpn85 TnpB nucleotide sequence

[0709]

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

[0711]

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

[0713]

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

[0715]

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

[0717]

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

[0719]

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

[0721]

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

[0723]

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

[0725]

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

[0727]

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

[0729]

[0730] SEQ ID NO:59 ISHhaal1 TnpB nucleotide sequence

[0731]

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

[0733]

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

[0735]

[0736] SEQ ID NO:62 ISC1sp11 TnpB nucleotide sequence

[0737]

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

[0739]

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

[0741]

[0742] SEQ ID NO:65 ISCath1 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:68 ISCno2 TnpB nucleotide sequence

[0749]

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

[0751]

[0752] SEQ ID NO:70 ISMba21 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> ISH1sp1 TnpB nucleotide sequence

[0831]

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

[0833]

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

[0835] GTATCAATATTCTGACCGAAGGTCTAAGACTACATTCTATGGGATTGGCTTAGATCCAATCATCAAGAACCGTAGGAACTACGGGGATAGCTTGGTAAATAAGAGACACCGCTGTTAGTAAAGAAATACGCTATCAAGTATGCTCTATTCCCAAGAAGCTCCCACTTCAAGCGTTAAGAACCGTCAGGTTTAGCTAAGTGGTGAGTAGTTCAC

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

[0837] GCCAGTTTAAATGCGCTGCGTGTGGTTATGAGGCGAACGCCGATGTGAACGGCGCTCGCAATATTTTAGCGGCGGGACACGCCGTTTTAGCCTGTGGAGGCGTGGTGCAATCAGGCCGCCCGATGAAGCAGGAACCCGGTACTGCGCGAGCAGCTATCACCCAGTAATGGGAATCCCCCTCCTGTGACGCGAAGCGCTAAGGTGGGGGAGGATGTCAA

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

[0839] GAATGTGGCTATAAGAATATAGAAGTTAAAAATCTAGGTCTAAGAGAATGGGTATGTCCCGAATGTGGAACACACCATCAAAGAGACCTTAATGCTTCAATAAACTTGGAAAAGCTGATAGCTTAAACTATTGGTATCCAACGAGGTCGGAACGCCCTTGTTAGCTTGGTTAATCTTACACCGTTAGGTGTGTTGCCCAAGAATCTTGGCATTTCAATGCCAAGTAGTTCAA

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

[0841] GTAATACATCACAAATGTGCTCTGGATGCGGTGAAATAGTTAAAAAAGAACTATCAGAGAGAATACACAGTTGTCCTTACTGTGGTCTTACTCTTGATAGAGATCACAATGCCGCTATTAACATTATGAGATTGGGATTACAATCTCTCCAGAATTCTGGTAGATGCCCGTCACTTCAGTGATGGGAGTAGTCAC

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

[0843] GTGCCGATGTGGATTTGAGAAACATCGTGATATTGTTGGGGCGATGAATATTCGCTACGCAACTGTGATTGATGGTAACAGTCAATCAGCCTAAGATGCTATACGCACTGTCTTAGGAGGGGCAATGAGATGCCCTCATCTTGAAGTCTGTTCAAAACAGAAATGGACTGCGAACGTTTAGTCATTCAAGAATCCCACCCGTCAATCCGTAAGGATTAGGGCTTGCGACTTTAGTCGTGGGAGTCTCAA

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

[0845] GCCGGACATGCCGTTGCATCAGCGGCAGTGGGTATGTCCAGCGTGTGGGGCAGGGCATGACCGCGACATTAACGCGGCCATGAATATCCAGCAACAAGGCATATTGGAATTAAAGGCGGCGGGACACGTCGTTTCTACCCATGGAGGCCAGCGTTAATCTGCCAATTCGGCAGTAGCGGCCTAAGAAGTGGGAAGCCTCGCCGCTTGCGGCGGGGAGCAGTCAC

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

[0847] GTGAAAGCCAAGCGGAGTTCCGGTGCCGCGCCTGCGGCCACCAGGCCAACGCCGACGTGAACGCCGCGAAGAACATCCTCGCCGCCGGGCTGGCGGTGACAGGGCGTGGAGACCTCGCCGTTGGGCGGTCTGTGAAGCGCCAACCACCCAAGGTAGAGGCAGCGTGAACGCGCCTCAGCCACGGGAATCCCCCGCCTTCAGGCGTGGGGAGGAAGTCAA <C

[0848] SEQ ID NO:118>ISCco1 gRNA scaffold 201ntGATAGAGTTTTTACTTGCGATAATTGTGGATATGTAGAACATAGAGATATAAATGCAAGTAAAAATATCTTGGATAGAGGCTTGAAGTCGTTTGGGCTAGGAACTAGCCTATTAGACTATAAACAAGAAGCCTTGCGTGTTAGCTGACATTGGTTGGCTTTAGTAAGGAAGCACCTACTTCTATAAGTAGGTGTGGTTCAC

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

[0850] GATCTATCCGTTCGCTCGGTCGTATGTCTAAATTGTGGTACAGAGCACGATAGAGACGAAAATGCTGCAAGAAACATAGAAAAAGTCGGGATAGGGCATTGCCACGACTATAAATGGACGCAGAGGGGGAGTAAGACTACTTCGGTAGCATCACCCAGTGAAGCGTCAAGAATCATCGCACTTTAGGCCGGTGAGTATGTCAA

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

[0852] GTTCTCACTGTGGTCATGTCTTAGAGAGTATTGATTTGTCTGTTCGGTCGTGGAGTTGTCCGTCCTGCCAGTCAGTGAACGAGAGAGACGAAAACGCCTCTAAGGTAATTTGTGCAGTCGGGGCATCGACTGTTGGGTTAGGTGATGTCAGACTGGCTACGCCAGCAATCGCTGTTTGAGCCTAGAATCCCCCGAATTATATTCGGGGGAGTATGTCAA

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

[0854] GAGATTACACGACAAAGAGATAGAAAGATTAAAAGAATTAAAGAAAAATGGATATAAATTAATATCCAGTATGGGGATATAAAATCTATATAGGTTTAGACATGAGCCTTATACTAATGTTAATCATTTATTAAAATGATAATTAGTGAAAGTCTTAGAGAAACTAACTAGTATTTATATGTTGTAATTTATTTGAAGTTTAAATAAATGAGAGTATAAAAGAAGTTAATGTATCTAAGAACCCCAAGGCTTTAGTCTTGGGAGTGTCAG

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

[0856] GACAGAGAATGGACTTGTCCTGATTGTAAAACAAAACATGATAGAGACATAAATGCCGCTATCAATATCAAGAAATTCGCTCTCATAGATCAAAATCTAATTGGATTGTAACACCTACGGAACGTGGGGAAGAGCTTGGGGACTTGCCCTCAATAGAGGGAAGAATGAACCAAGAAGCAACTCAGTCTTTAGCTGAGTGGTAGTTCAC

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

[0858] GAGATGCCATTACATAAGCGCATCTGGCAGTGTCCAGAGTGTGGAGTTGAACATGACCGCGATATCAACGCGGCGCTCAACATCAAACGCAAGGGCATACTGGAATTACAAGCGGCGGGACTCGTCGTTTCTGCCCATGGAGGCCAGCATAAATCCGTCACACAGACGGTTGCGGCTTAAGAAGTGGGAAGCCTCGCCCGACAGGGCGGGGAGCAGTCAC

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

[0860] GTAAATTTGTGTGTCAGGCATGCGGATATACAGCGAACGCTGATGTAAACGGCGCTCGTAACATTTTAGCGGCGGGGCACGCCGTTCTTGCCTGTGGAGGGATGGTGCAGTCAGGCCGCCCGTCTGAAACAGGAACCAGACGCTGAGCAATCAGTCGTCACCCACTAATGGGAATTCCCCACCTGAGTCACGAAGTGCGAAGGTGGGGGAGGATGTCAA

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

[0862] GCGCTGGCACAGCTGCCCTTGCGGCGCGTCCTGTTCACGGGACGAGAACGCCGCTCGGGTGCTGTTGGCTTGGCTGGAGCGGAGTTTATCCGGTCGGGAACCGGCCGATGCGTGGAGGGAGGTCAGACCGGGTCATCCTTTGGATGAATCGGCACTCCCGTCGAAGCGCGAAACTCACGCCATAGCGTAAGCTTGGCGGGAGTAGTTCAT

[0863] SEQ ID NO:126> ISkpn69 gRNA scaffold 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 scaffold 201nt

[0872] GAATGTGGCTACAAAGAGAACGCCGACATAAACGGCGCTCGTAATATTTTAGCGGCAGGACATGCCGTTTTAGCCTGTGAAGTGAACGGTGCATCAATGCCGTCAGCAGCAGGAACCAAGCGACTGAGCAATCAGTTAGCCACCCAGtaaTGGGAATCCCCCACCTGAATCACGTAGTGTGAAGGTGGGGGAGGATGTCAA

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

[0874] GAAAACCTAGATTTGTCAATTAGACGCTGGCGTTGTCCTTCATGTCAATCTGAAAATGATCGGGATGGAAACGCTAGTTTTGTTGAATATTAAAAGAGTCGGGAGTTCGACTCTAGGCGTAGGAGATGTTAGACAGTCCCAGACTGCAATCTCTGTCtgaTGCCTGAATCCCCCGTTTTCAAAACGGGGGAGTGGCTCAA

[0875] SEQ ID NO:132 > ISAba30 gRNA scaffold 204 nt GCATAGAGTGCGGGTTCGAGGGTAATGCCGATGTGGTCGGCGCGATCAATGTACTAAGGGCGGGGCACGCCCGGTTAGCCTGTGAAGTGAGCGATGCAGTAGGGTCGCCAGCAGCAGGAACCCACCGAAGCGACTCAGACCAGGCTCATTGCCGCGTCtgaGCGCCGTAGGAATCTCCGGCCTTCAGGCCGGGGAGGATGTCAA

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

[0877] GAATGTGGATTCGAGAGCGACAGGGACGTCAATGCGGCAATCAATATCAAACATGAGGGCATGAAACGATTAGCAATAGCCtaaCTTGTCCTCGAACCGTGGGACACACGGGGATCGCTCAGTCAACTTCCCGTCATGAGATGGGATTACCTGAGAAGCCCCCACCTCTAAGCGAAGCGTAGGTGGTGGGAGCATGTCAC

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

[0879] GTCAATGCAGCAAGAAATATACTGGCTGAGGGACTCAGGATAAGAGCTCTGACTCCGGGGTCTtagCAATACAGAAGCAAACAGGAACCGCAGGAATTGCGGGGGTAGCTTGGTAAACAAGAGAAACCTCTGCCGGCAAAGAAATAAGCCGGTAAGTATGCTCTGTTCCCAAGAATCTCGTGACTTTAGTCATGAGAGTTTCAA

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

[0881] GATAGAGATTACAATTCAGCACTCAATATCAAAAAAGAAGGCATTCGCTTATTAGCAAGTGCCtaaAGAAATAAGAGATATAAACTCTTGGAACAAGAGGGTTAGCTTGGTCAATTTCGTCAGCTACCAAAAGTGGCGATTACCCAAGAAGCCCCCACTTCAAGCAACCCGCAAGGGTGGTAAGTGGTGGGAGTAGTTCAC

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

[0883] GCAAAGCACGATAGAGACACCAACGCAAGCATAACCATCTTAAATAAAGGGCTGTCAATAGCCtgaCATATATAAAATACCGTAGGGCATACGGGAATTTACGCTTGTGGACACTGTGTAAGACGAGCCAACAAGGTAACTTGTTAGGAACGCAGTAGTGGTTGAAGCAAGAATCCCACGACTTTAGTCGTGTGGAGTGTCAA

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

[0885] GAATGCCGCTATCAATATCTTACGTGAGGGATTACGACTACTGACTAAAGTAGCCtaaAACTAAATATTACAGTAGGGATGGAATAGCCCAAACTCATACGCCTGTGGACACTGTGTAAGACGAGCCGTTACGTATCATCGTAGCCAGCGCAGTAGTGGTTGAAGCAGGAAGCTCGGTTACTTGTAACCGAGTAGTTCAC

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

[0887] GTTCTTGCGGTTTTGTATTAGATCGTGATCACAATTCAGCAATCAATATCAAAAATGAAGGATTACGCATATTAGCAtaaAGAATAATAACTTTCGGAACGAGAGGGTAAGCTCGGTCAATTTCTTATCGTTAGATAGGATTACCCGAGAAGCCCCCACTTCAAGCGACCCGCAAGGGTGGTAAGTGGAGGGTAGTTCAC

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

[0889] GTCTGAGCGAGTCTATTCTTGTATATGCGGTGTAAATCTCGATAGAGATTATAACGCAGCTATCAATATCAAAAATGAAGCAATACGCCTATTGGCGTTAGTAtagATAGCAATAAAATAACCCTTGGAACAAGGGAGTTAGCTCGGCTGTCTTAGGACGAATTCGTTAGCTACCAAAAGTAACGACTAACCGAGAAGCCCCCACTTCAAACAGTCCGTTAGGACGTTAAGTGGCGGGTAGTTCAC

[0890] SEQ ID NO:140 mRFP coding sequence

[0891] ATGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCCAGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGATGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTCAAGACCACCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAAGACCGACATCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCGCCGAGGGCCGCCACTCCACCGGCGCC

[0892] SEQ ID NO:141 GFP coding sequence

[0893] GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

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

[0895] GGAGGTGAAAAGGCCCTCAT

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

[0897] GTGCCCAACAGCTTGTATGA

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

[0899] TATGGAATACCCTGTATGAA

[0900] SEQ ID NO: 145 Target sequence in hHPRT of ISDgel0

[0901] AGCCCCCCTTGAGCACACAG

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

[0903] ATTCTCCAGGAGGAAGAACC

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

[0905] GTTATCCATAAAAGAATTCC

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

[0907] GTAATACAATGTTCTTGTCA

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

[0909] GTTAGGATTAGGTGGGCTCA

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

[0911] CAGGTCCTCATAGGTCGAGT

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

[0913] TCAAGTGCTGTTTCAACACT

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

[1026] SEQ ID NO:208

[1027] GAACCGCAGGAATTGCGGGGGTAGCTTGGTAAACAAGAGAAACCTCTGCCGGCAAAGAAATAAGCCGGTAAGTATGCTCTGTTCCAAGAATCTCGTGACTTTAGTCATGAGAGTTTCAA

[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 Target sequence for screening TnpB

[1091] GCTCGGAGATCATCATTGCG

Claims

1. A recombinant gene editing system comprising - a TnpB polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding a TnpB polypeptide or a functional fragment thereof, and - a guide RNA (gRNA), or a polynucleotide comprising a nucleotide sequence encoding a gRNA, wherein the gRNA comprises a targeting region capable of hybridizing with a nucleotide sequence of interest and a backbone region capable of binding to the TnpB polypeptide or a functional fragment thereof, wherein the TnpB polypeptide is derived from a microorganism selected from the group consisting of Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas media, Thermobifida fusca, Campylobacter coli, Synechococcus sp. JA-3-3Ab, Thermosynechococcus elongatus, Nostoc sp. PCC 7120, Clostridium botulinum C-Stockholm phage c-st, Methanosarcina acetivorans, Escherichia coli, 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-guided endonuclease activity.

2. The recombinant gene editing system of claim 1, wherein the TnpB polypeptide comprises an amino acid sequence that is at least 70% identical to 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.

3. The recombinant gene editing system of claim 1 or 2, comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises a nucleotide sequence encoding a TnpB polypeptide or a functional fragment thereof, and the second polynucleotide comprises a nucleotide sequence encoding a gRNA.

4. The recombinant gene editing system of any one of claims 1 to 3, wherein the TnpB polypeptide or a functional fragment thereof recognizes a transposon-associated motif (TAM) adjacent to a nucleotide sequence of interest and has endonuclease activity.

5. The recombinant gene editing system of any one of claims 1-4, wherein the TnpB polypeptide comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:7, 18, 21, 23 or 24.

6. The recombinant gene editing system of claim 4, wherein the TAM consists of 4 consecutive nucleotides.

7. The recombinant gene editing system of any one of claims 1-6, further comprising a heterologous polynucleotide.

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

9. A composition comprising: - a recombinant TnpB polypeptide or a functional fragment thereof, - a target double-stranded DNA comprising a nucleotide sequence of interest and a TAM recognized by said TnpB polypeptide; and - a recombinant guide RNA (gRNA) comprising a targeting region capable of hybridizing with the nucleotide sequence of interest and a backbone region capable of binding to the TnpB polypeptide or a functional fragment thereof, wherein the TnpB polypeptide is derived from a microorganism selected from the group consisting of Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas intermedia, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc PCC 7120, Clostridium botulinum type C C-Stockholm phage c-st, Methanosarcina acetophilus, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Raoultia ornithine, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Bacillus pallidum, and Bacillus thuringiensis, and wherein the TnpB polypeptide has RNA-guided endonuclease activity.

10. The composition of claim 9, wherein the TnpB polypeptide comprises an amino acid sequence that is at least 70% identical to 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.

11. The composition of claim 9 or 10, wherein the TnpB polypeptide or a functional fragment thereof recognizes TAM adjacent to a nucleotide sequence of interest and has endonuclease activity.

12. The composition of any one of claims 9-11, wherein the TnpB polypeptide comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 7, 18, 21, 23 or 24.

13. The composition of claim 11, wherein the TAM consists of 4 consecutive nucleotides.

14. The composition of any one of claims 9-13, further comprising a heterologous polynucleotide.

15. The composition of claim 14, wherein the heterologous polynucleotide is an expression cassette, a transgene, a donor DNA, or a polynucleotide modification template.

16. 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 - a TnpB polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding a TnpB polypeptide or a functional fragment thereof, and - a guide RNA (gRNA) comprising a targeting region capable of hybridizing with a nucleotide sequence in a polynucleotide of interest and a backbone region capable of binding to the TnpB polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding a gRNA, wherein the TnpB polypeptide is derived from a microorganism selected from the group consisting of Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas intermedia, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc PCC 7120, Clostridium botulinum type C C-Stockholm phage c-st, Methanosarcina acetophilus, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Raoultia ornithine, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Bacillus pallidum, and Bacillus thuringiensis, and wherein the TnpB polypeptide has RNA-guided endonuclease activity.

17. The method of claim 16, wherein the TnpB polypeptide comprises an amino acid sequence that is at least 70% identical to 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.

18. The method of claim 16 or 17, wherein the gene editing system comprises a TnpB polypeptide or a functional fragment thereof and a gRNA, or a first polynucleotide comprising a nucleotide sequence encoding a TnpB polypeptide or a functional fragment thereof and a second polynucleotide comprising a nucleotide sequence encoding a gRNA.

19. The method of any one of claims 16 to 18, wherein the TnpB polypeptide or a functional fragment thereof recognizes TAM adjacent to a nucleotide sequence of interest and has endonuclease activity.

20. The method of any one of claims 16-19, wherein the TnpB polypeptide comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 7, 18, 21, 23 or 24.

21. The method of claim 19, wherein the TAM consists of 4 consecutive nucleotides.

22. The method of any one of claims 16-21, wherein the gene editing system further comprises a heterologous polynucleotide.

23. The method of claim 22, wherein the heterologous polynucleotide is an expression cassette, a transgene, a donor DNA, or a polynucleotide modification template.

24. 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 - a TnpB polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding a TnpB polypeptide or a functional fragment thereof, and - a guide RNA (gRNA) comprising a targeting region capable of hybridizing with a portion of a genomic sequence and a backbone region capable of binding to the TnpB polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding a gRNA, wherein the TnpB polypeptide is derived from a microorganism selected from the group consisting of Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas intermedia, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc PCC 7120, Clostridium botulinum type C C-Stockholm phage c-st, Methanosarcina acetophilus, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Raoultia ornithine, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Bacillus pallidum, and Bacillus thuringiensis, and wherein the TnpB polypeptide has RNA-guided endonuclease activity.

25. The method of claim 24, wherein the TnpB polypeptide comprises an amino acid sequence at least 70% identical to 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.

26. The method of claim 24 or 25, wherein the recombinant gene editing system comprises a first polynucleotide and a second polynucleotide, the first polynucleotide comprises a nucleotide sequence encoding a TnpB polypeptide or a functional fragment thereof, and the second polynucleotide comprises a nucleotide sequence encoding a gRNA.

27. The method of any one of claims 24-26, wherein the TnpB polypeptide or a functional fragment thereof recognizes TAM adjacent to a nucleotide sequence of interest and has endonuclease activity.

28. The method of any one of claims 24-27, wherein the TnpB polypeptide comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 7, 18, 21, 23 or 24.

29. The method of claim 27, wherein the TAM consists of 4 consecutive nucleotides.

30. The method of any one of claims 24-29, wherein the gene editing system further comprises a heterologous polynucleotide.

31. The method of claim 30, wherein the heterologous polynucleotide is an expression cassette, a transgene, a donor DNA, or a polynucleotide modification template.

32. The method of any one of claims 24-31, wherein the cell is a prokaryotic cell or a eukaryotic cell.

33. A modified TnpB polypeptide comprising a modification in a DDE motif compared to a parent TnpB polypeptide, wherein the parent polypeptide has RNA-guided endonuclease activity, and wherein the modified TnpB loses the activity of cleaving double-stranded DNA.

34. The modified TnpB polypeptide of claim 33, wherein at least one amino acid in the DDE motif is substituted with alanine, and the amino acid corresponding to N31 of SEQ ID NO:7 is substituted with alanine.

35. The modified TnpB polypeptide of claim 33 or 34, wherein the parent TnpB polypeptide is derived from a microorganism selected from the group consisting of Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas intermedius, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcuse longatus, Nostoc sp. PCC 7120, Clostridium botulinum type C C-Stockholm phage c-st, Methanosarcina acetophilus, Escherichia coli, Salmonella enterica, Halorubrum halophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Raoultia ornithinelyticus, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp. CY-G, Clostridium vitabionis, Bacillus aeruginosa, and Bacillus thuringiensis.

36. The modified TnpB polypeptide of claim 33, wherein the modified TnpB polypeptide comprises an amino acid sequence that is at least 70% identical to 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.

37. A recombination system comprising - The modified TnpB polypeptide or a functional fragment thereof according to any one of claims 33 to 36, or a polynucleotide comprising a nucleotide sequence encoding the modified TnpB polypeptide or a functional fragment thereof, and - 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 polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding a gRNA.

38. The recombinant system of claim 37, comprising a first polynucleotide and a second polynucleotide, the first polynucleotide comprising a nucleotide sequence encoding a modified TnpB polypeptide or a functional fragment thereof, and the second polynucleotide comprising a nucleotide sequence encoding a gRNA.

39. The recombination system of claim 37 or 38, wherein the gRNA further comprises one or more protein binding domains.

40. A method for modifying a genomic sequence in a cell, comprising the steps of introducing into the cell the recombination system of any one of claims 37-39 and a gene editing system targeting the genomic sequence, wherein a nucleotide sequence of interest is proximal to the genomic sequence.

41. A fusion polypeptide comprising a TnpB polypeptide or a functional fragment thereof or a disarmed variant thereof fused to a fusion partner, wherein the TnpB polypeptide is derived from a microorganism selected from the group consisting of Enterococcus faecium, Aeromonas salmonicida, Clostridium perfringens, Methanosarcina mazei, Bacillus cereus, Aeromonas intermedia, Thermobifida fusca, Campylobacter coli, Synechococcus JA-3-3Ab, Thermosynechococcus elongatus, Nostoc sp. PCC 7120, Clostridium botulinum type C C-Stockholm phage c-st, Methanosarcina acetophilus, Escherichia coli, Salmonella enterica, Halorubrumhalophilum, Klebsiella pneumoniae, Deinococcus geothermalis, Acinetobacter baumannii, Raoultia ornithinelyticus, Anoxybacillus amylolyticus, Youngiibacter multivorans, Cytobacillus sp.CY-G, Clostridium vitabionis, Bacillus pallidum and Bacillus thuringiensis, and has RNA-guided endonuclease activity, and wherein the disarmed variant is the modified TnpB polypeptide of any one of claims 33-36.

42. The fusion polypeptide of claim 41, wherein the TnpB polypeptide comprises an amino acid sequence that is at least 70% identical to 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.

43. The fusion polypeptide of claim 41 or 42, wherein the TnpB polypeptide has the activity of cleaving double-stranded DNA.

44. The fusion polypeptide of any one of claims 41-43, wherein the TnpB polypeptide or a functional fragment thereof recognizes TAM adjacent to a nucleotide sequence of interest and has endonuclease activity.

45. The fusion polypeptide of claim 44, wherein the TAM consists of 4 consecutive nucleotides.

46. ​​A gene editing system comprising - A fusion polypeptide according to any one of claims 41 to 44, or a polynucleotide comprising a nucleotide sequence encoding said fusion polypeptide, and - 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 polypeptide or a functional fragment thereof, or a polynucleotide comprising a nucleotide sequence encoding a gRNA.

47. The gene editing system of claim 46, comprising a first polynucleotide and a second polynucleotide, the first polynucleotide comprising a nucleotide sequence encoding the fusion polypeptide, and the second polynucleotide comprising a nucleotide sequence encoding a gRNA.

48. The gene editing system of claim 46 or 47, further comprising a heterologous polynucleotide.

49. The gene editing system of claim 48, wherein the heterologous polynucleotide is an expression cassette, a transgene, a donor DNA, or a polynucleotide modification template.

50. A method for modifying a genomic sequence in a eukaryotic cell, comprising the step of introducing the gene editing system of any one of claims 46-49 into the eukaryotic cell, wherein the gRNA comprises a targeting region capable of hybridizing with a portion of the genomic sequence.

51. A method for screening a TnpB polypeptide having double-stranded DNA cleavage activity, comprising the following steps: - providing a candidate TnpB polypeptide from a microorganism; - providing a gRNA comprising a targeting region and a framework region, wherein the framework region comprises 100-350 nucleotides before the 3' end of the IS, and the IS naturally comprises a nucleotide sequence encoding the TnpB polypeptide; - providing a target DNA comprising a nucleotide sequence that hybridizes with the nucleotide sequence of the targeting region and a TAM recognized by the TnpB polypeptide, wherein the TAM consists of 4 or 5 consecutive nucleotides adjacent to the 5' end of the IS; - contacting the TnpB polypeptide with the gRNA and the target DNA; and - Detection of cleavage on target DNA.

52. The method of claim 51, wherein the TnpB polypeptide is provided as a first polynucleotide comprising a first nucleotide sequence encoding the TnpB polypeptide.

53. The method of claim 51 or 52, wherein the gRNA is provided as a second polynucleotide comprising a second nucleotide sequence encoding the gRNA.

54. The method of claim 52 or 53, wherein the first and second polynucleotides are provided in a first plasmid.

55. The method of claim 52, wherein the first nucleotide sequence is operably linked to a first promoter.

56. The method of claim 53, wherein the second nucleotide sequence is operably linked to a second promoter.

57. The method of any one of claims 51-56, wherein the target DNA is provided in a second plasmid.

58. The method of claim 57, wherein contacting the TnpB polypeptide with the gRNA and the target DNA comprises introducing a first and a second plasmid into a host cell comprising the target DNA.

59. The method of any one of claims 51-58, wherein the TnpB polypeptide comprises an N-terminal HTH domain, a central domain, a C-terminal zinc finger domain, and a DDE motif.

60. A fusion polypeptide comprising the modified TnpB polypeptide of any one of claims 33-36 fused to a fusion partner.

61. The fusion polypeptide of claim 60, wherein the fusion partner is a polypeptide that provides an activity that indirectly increases transcription by acting directly on the target DNA or a polypeptide associated with the target DNA (e.g., a histone).

62. The fusion polypeptide of claim 60, wherein the fusion partner is a polypeptide that provides methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity.

63. The fusion polypeptide of claim 60, wherein the fusion partner is a polypeptide that directly provides for increased transcription of a target nucleic acid.

64. The fusion polypeptide of claim 63, wherein the fusion partner is a transcriptional activator or a fragment thereof, a protein or a fragment thereof that recruits a transcriptional activator, a small molecule / drug responsive transcriptional regulator.

65. The fusion polypeptide of claim 60, wherein the fusion partner is another polypeptide or domain that produces a double-strand break.

66. The fusion polypeptide of claim 60, wherein the fusion partner is a polypeptide that directs the editing of single or multiple bases in a polynucleotide sequence.

67. The fusion polypeptide of claim 66, wherein the fusion partner is a site-specific deaminase that can change the identity of a nucleotide, for example from CG to TA or from AT to GC.

68. The fusion polypeptide of claim 66, wherein the fusion partner is a deaminase such as cytidine deaminase, adenine deaminase, APOBEC1, APOBEC3A, BE2, BE3, BE4 or ABE.

69. The fusion polypeptide of claim 66, wherein the fusion partner comprises a base editing repair inhibitor and a glycosylase inhibitor.

70. The fusion polypeptide of claim 60, wherein the fusion partner can be a Cas endonuclease or another TnpB endonuclease as described in the present disclosure.

71. The fusion polypeptide of claim 60, wherein the fusion partner is a nuclear localization sequence (NLS).

72. A recombinant adeno-associated virus (rAAV), the genome of which comprises a first expression cassette encoding the fusion polypeptide of any one of claims 41-45 and 60-71.

73. The rAAV of claim 72, wherein the genome comprises a second expression cassette encoding a guide RNA (gRNA), wherein the guide RNA (gRNA) comprises a targeting 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.

74. The rAAV of claim 71 or 72, wherein the first expression cassette comprises less than about 4,700 nucleotides, less than about 4,600 nucleotides, less than about 4,500 nucleotides, less than about 4,400 nucleotides, less than about 4,300 nucleotides, less than about 4,200 nucleotides, less than about 4,100 nucleotides, or less than about 4,000 nucleotides.

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