Systems and methods for transposing cargo nucleotide sequences

Abstract

The present disclosure provides systems and methods for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid. These systems and methods can include: a double-stranded nucleic acid comprising the cargo nucleotide sequence, where the cargo nucleotide sequence interacts with a recombinase complex; an effector complex, the effector complex comprising an effector and at least one engineered guide polynucleotide, the at least one engineered guide polynucleotide hybridizing to the target nucleic acid; and the recombinase recognition complex, wherein the recombinase recognition complex recruits the cargo nucleotide to the target nucleic acid site.

Description

Cross-referencing

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 501,227, filed May 10, 2023, and U.S. Provisional Patent Application No. 63 / 514,768, filed July 20, 2023, each of which is incorporated herein by reference in its entirety. sequence list

[0002] This application contains a sequence list that has been electronically submitted in XML format, and the sequence list is hereby incorporated in its entirety by reference. The XML copy created on May 3, 2024, is named MTG-026WO_SL.xml and has a size of 1,536,000 bytes. Background Technology

[0003] Cas enzymes and their associated clustered regularly spaced short palindromic repeats (CRISPR)-guided ribonucleic acid (RNA) appear to be a ubiquitous component of the prokaryotic immune system (approximately 45% of bacteria and 84% of archaea) used to protect such microorganisms from non-self nucleic acids, such as infectious viruses and plasmids, via CRISPR-RNA-guided nucleic acid cleavage. While the deoxyribonucleic acid (DNA) elements encoding CRISPR RNA elements may be relatively conserved in structure and length, their CRISPR-associated (Cas) proteins are highly diverse, containing a variety of nucleic acid interaction domains. Although CRISPR DNA elements were observed as early as 1987, the programmable endonuclease cleavage capability of CRISPR / Cas complexes has only recently been recognized, leading to the use of recombinant CRISPR / Cas systems in a variety of DNA manipulation and gene editing applications. Summary of the Invention

[0004] In some aspects, this disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site, the system comprising: a first double-stranded nucleic acid comprising a cargo nucleotide sequence configured to interact with a Tn7-type transposase complex; a Cas effector complex comprising two class V Cas effectors and an engineered guide polynucleotide configured to hybridize with the target nucleotide sequence; and a Tn7-type transposase complex configured to bind to the Cas effector complex, wherein the Tn7-type transposase complex comprises a TnsB subunit. In some embodiments, the cargo nucleotide sequence is flanked by a left-side transposase recognition sequence and a right-side transposase recognition sequence. In some embodiments, the system further comprises a second double-stranded nucleic acid containing the target nucleic acid site. In some embodiments, the system further comprises a PAM sequence compatible with the Cas effector complex adjacent to the target nucleic acid site. In some embodiments, the PAM sequence is located at the 3' position of the target nucleic acid site.

[0005] In some aspects, this disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a Cas effector complex comprising two types V Cas effectors, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide that hybridizes to the target nucleic acid site; a Tn7 transposase complex that binds to the Cas effector complex and comprises a TnsB component, a TnsC component, and a TniQ component, and an accessory protein comprising a sequence having at least 70% sequence identity with any one of SEQ ID NO: 228-230 and 235-249; and a double-stranded nucleic acid that interacts with the Tn7 transposase complex and comprises the cargo nucleotide sequence.

[0006] In some embodiments, the Cas effector complex is non-covalently bound to the Tn7 transposase complex. In some embodiments, the Cas effector complex is covalently linked to the Tn7 transposase complex. In some embodiments, the Cas effector complex is fused to the Tn7 transposase complex.

[0007] In some embodiments, the cargo nucleotide sequence is flanked by a left-sided transposase recognition sequence and a right-sided transposase recognition sequence recognized by the Tn7 type transposase complex. In some embodiments, the left-sided transposase recognition sequence comprises a sequence having at least 80% identity with any one of SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the right-sided transposase recognition sequence comprises a sequence having at least 80% identity with any one of SEQ ID NO: 8, 10, 39-44, 77, 79, and 93.

[0008] In some embodiments, the target nucleic acid includes a PAM sequence compatible with the Cas effector complex. In some embodiments, the PAM sequence includes SEQ ID NO: 31. In some embodiments, the PAM sequence is located at approximately 50 to approximately 70 base pairs from the target nucleic acid site. In some embodiments, the PAM sequence is located at the 3' position of the target nucleic acid site. In some embodiments, the PAM sequence is located at the 5' position of the target nucleic acid site.

[0009] In some embodiments, the type 2 V Cas effector is the Cas12k effector. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least 80% identity with any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220.

[0010] In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least 90% identity with any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence of any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220.

[0011] In some embodiments, the TnsB component comprises a polypeptide having a sequence having at least 80% identity with any of SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide having a sequence having at least 90% identity with any of SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide having a sequence having any of SEQ ID NO: 2, 13, 17, and 65.

[0012] In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently comprising a sequence having at least 80% identity with any one of SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently comprising a sequence having at least 90% identity with any one of SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently comprising a sequence of any one of SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111.

[0013] In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% identity with any one of SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222, consisting of at least about 46-80 consecutive nucleotides. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944.

[0014] In some embodiments, the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity with any of SEQ ID NO: 187-189. In some embodiments, the small prokaryotic ribosomal protein subunit S15 is encoded by a sequence having at least 80% sequence identity with any of SEQ ID NO: 181-183.

[0015] In some embodiments, the type V Cas effector and the Tn7 transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0016] In some embodiments, the accessory protein is ClpX, which comprises a sequence having at least 80% sequence identity with any of SEQ ID NO: 235-249.

[0017] In another aspect, this disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site, the system comprising: a Cas effector complex including two types V Cas effectors, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide that hybridizes to the target nucleic acid site; a Tn7 transposase complex that binds to the Cas effector complex and includes a functional domain (FD)-TniQ fusion and an accessory protein; and a double-stranded nucleic acid that interacts with the Tn7 transposase complex and includes the cargo nucleotide sequence.

[0018] In some embodiments, the Cas effector complex is non-covalently bound to the Tn7 transposase complex. In some embodiments, the Cas effector complex is covalently linked to the Tn7 transposase complex. In some embodiments, the Cas effector complex is fused to the Tn7 transposase complex.

[0019] In some respects, this disclosure provides the said functional structural domain (FD), including sequences having at least 70% identity with any of SEQ ID NO: 257-307 and 1138-1242.

[0020] In some embodiments, the cargo nucleotide sequence is flanked by a left-sided transposase recognition sequence and a right-sided transposase recognition sequence recognized by the Tn7 type transposase complex. In some embodiments, the left-sided transposase recognition sequence comprises a sequence having at least 80% identity with any one of SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the right-sided transposase recognition sequence comprises a sequence having at least 80% identity with any one of SEQ ID NO: 8, 10, 39-44, 77, 79, and 93.

[0021] In some embodiments, the target nucleic acid includes a PAM sequence compatible with the Cas effector complex. In some embodiments, the PAM sequence includes SEQ ID NO: 31. In some embodiments, the PAM sequence is located at approximately 50 to approximately 70 base pairs from the target nucleic acid site. In some embodiments, the PAM sequence is located at the 3' position of the target nucleic acid site. In some embodiments, the PAM sequence is located at the 5' position of the target nucleic acid site.

[0022] In some embodiments, the type 2 V Cas effector is the Cas12k effector. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least 90% identity with any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence of any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220.

[0023] In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% identity with any one of SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222, consisting of at least about 46-80 consecutive nucleotides. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944.

[0024] In some embodiments, the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity with any of SEQ ID NO: 187-189. In some embodiments, the small prokaryotic ribosomal protein subunit S15 is encoded by a sequence having at least 80% sequence identity with any of SEQ ID NO: 181-183. In some embodiments, the type V Cas effector and the Tn7 transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0025] In some embodiments, the accessory protein comprises a sequence having at least 70% sequence identity with any of SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein is ClpX, which comprises a sequence having at least 80% sequence identity with any of SEQ ID NO: 235-249.

[0026] In another aspect, this disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site, the system comprising: a Cas effector complex comprising two types of V Cas effectors and an engineered guide polynucleotide, the engineered guide polynucleotide hybridizing to the target nucleic acid site, wherein the Cas effector complex comprises a polypeptide having at least 80% sequence identity with any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220; a Tn7 transposase complex bound to the Cas effector complex and comprising a TnsB component, a TnsC component, and a TniQ component, and an accessory protein, wherein the TnsB component, the TnsC component, or the TniQ component comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 2-4, 13-15, 17-19, 65-67, and 109-111, and the accessory protein ... the accessory protein comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 2-4, 13-15, 17-19, 65-67, and 109-111, the accessory protein comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 2-4, 13-15, 17-19, 65-67, and 109-111, the accessory protein comprising a sequence having at least 80% sequence identity with any one of SEQ ID The sequence having at least 80% sequence identity among NO:228-230 and 235-249; and a double-stranded nucleic acid that interacts with the Tn7 transposase complex and includes the cargo nucleotide sequence.

[0027] In some embodiments, the Cas effector complex is non-covalently bound to the Tn7 transposase complex. In some embodiments, the Cas effector complex is covalently linked to the Tn7 transposase complex. In some embodiments, the Cas effector complex is fused to the Tn7 transposase complex.

[0028] In some embodiments, the cargo nucleotide sequence is flanked by a left-sided transposase recognition sequence and a right-sided transposase recognition sequence recognized by the Tn7 type transposase complex. In some embodiments, the left-sided transposase recognition sequence comprises a sequence having at least 80% identity with any one of SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the right-sided transposase recognition sequence comprises a sequence having at least 80% identity with any one of SEQ ID NO: 8, 10, 39-44, 77, 79, and 93.

[0029] In some embodiments, the target nucleic acid includes a PAM sequence compatible with the Cas effector complex. In some embodiments, the PAM sequence includes SEQ ID NO: 31. In some embodiments, the PAM sequence is located at approximately 50 to approximately 70 base pairs from the target nucleic acid site. In some embodiments, the PAM sequence is located at the 3' position of the target nucleic acid site. In some embodiments, the PAM sequence is located at the 5' position of the target nucleic acid site.

[0030] In some embodiments, the type 2 V Cas effector is the Cas12k effector. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least 90% identity with any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220.

[0031] In some embodiments, the type 2 V-type Cas effector comprises a polypeptide containing a sequence of any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220.

[0032] In some embodiments, the TnsB component, the TnsC component, or the TniQ component comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 2-4, 13-15, 17-19, 65-67, and 109-111. In some embodiments, the TnsB component, the TnsC component, or the TniQ component comprises a sequence of any one of SEQ ID NO: 2-4, 13-15, 17-19, 65-67, and 109-111.

[0033] In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% identity with any one of SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222, consisting of at least about 46-80 consecutive nucleotides. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944.

[0034] In some embodiments, the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity with any of SEQ ID NO: 187-189. In some embodiments, the small prokaryotic ribosomal protein subunit S15 is encoded by a sequence having at least 80% sequence identity with any of SEQ ID NO: 181-183.

[0035] In some embodiments, the type V Cas effector and the Tn7 transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0036] In some embodiments, the accessory protein is ClpX, which comprises a sequence having at least 90% sequence identity with any of SEQ ID NO: 235-249.

[0037] In some aspects, this disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site, the system comprising: a Cas effector complex that hybridizes to the target nucleic acid site and includes: i) two types of V Cas effectors, the two types of V Cas effectors including a sequence having at least 80% sequence identity with any one of SEQ ID NO: 1, 81, 82, 83 and 85; and ii) an engineered guide polynucleotide that has at least 80% sequence identity with any one of SEQ ID NO: 5, 6, 45-63, 68-75, 96-103, 123-140 and 754-944; a Tn7 transposase complex that binds to the Cas effector complex and includes a TnsB component, a TnsC component and a TniQ component, and an accessory protein, the TnsB component, the TnsC component or the TniQ component including a sequence having at least 80% sequence identity with any one of SEQ ID NO: 1, 81, 82, 83 and 85; and ii) an engineered guide polynucleotide that hybridizes to the target nucleic acid site and includes: i) two types of V Cas effectors, the two types of V Cas effectors including a sequence having at least 80% sequence identity with any one of SEQ ID NO: 1, 81, 82, 83 and 85; and ii) a Tn7 transposase complex that hybridizes to the target nucleic acid site and includes: i) ... The accessory protein comprises a sequence having at least 80% identity with any of 2-4, the accessory protein comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 228-230 and 235-249; and a double-stranded nucleic acid that interacts with the Tn7 type transposase complex and comprises, in 5' to 3' order: i) a left transposase recognition sequence comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 9, 11, 36, 37 and 38; ii) the cargo nucleotide sequence; and ii) a right transposase recognition sequence comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 8, 39-44 and 93.

[0038] In some aspects, this disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site, the system comprising: a Cas effector complex that hybridizes to the target nucleic acid site and comprising: i) two types of V Cas effectors, the two types of V Cas effectors comprising a sequence having at least 80% sequence identity with SEQ ID NO: 12; and iii) an engineered guide polynucleotide that has at least 80% identity with any one of SEQ ID NO: 32, 102, 104, and 107; a Tn7 transposase complex that binds to the Cas effector complex and comprises a TnsB component, a TnsC component, and a TniQ component, and an accessory protein, the TnsB component, the TnsC component, or the TniQ component comprising a sequence having at least 80% identity with any one of SEQ ID NO: 13-15, and the accessory protein comprising a sequence having at least 80% identity with any one of SEQ ID NO: 12; and iii) an engineered guide polynucleotide that has at least 80% sequence identity with any one of SEQ ID NO: 12; The sequence having at least 80% sequence identity among any of 228-230 and 235-249; and a double-stranded nucleic acid that interacts with the Tn7 type transposase complex and comprises, in 5' to 3' order: i) a left transposase recognition sequence comprising a sequence having at least 80% sequence identity with SEQ ID NO: 76; ii) the cargo nucleotide sequence; and iii) a right transposase recognition sequence comprising a sequence having at least 80% identity with SEQ ID NO: 77.

[0039] In some aspects, this disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site, the system comprising: a Cas effector complex that hybridizes to the target nucleic acid site and comprising: i) two types of V Cas effectors, the two types of V Cas effectors comprising a sequence having at least 80% sequence identity with SEQ ID NO: 16; and ii) an engineered guide polynucleotide that has at least 80% identity with any one of SEQ ID NO: 33, 103, 105, and 108; a Tn7 transposase complex that binds to the Cas effector complex and comprises a TnsB component, a TnsC component, and a TniQ component, and an accessory protein, the TnsB component, the TnsC component, or the TniQ component comprising a sequence having at least 80% identity with any one of SEQ ID NO: 17-19, and the accessory protein comprising a sequence having at least 80% identity with any one of SEQ ID NO: 16; and ii) an engineered guide polynucleotide that has at least 80% sequence identity with any one of SEQ ID NO: 16; The sequence having at least 80% sequence identity among any one of 228-230 and 235-249; and a double-stranded nucleic acid that interacts with the Tn7 type transposase complex and comprises, in 5' to 3' order: i) a left transposase recognition sequence comprising a sequence having at least 80% sequence identity with SEQ ID NO: 78; ii) the cargo nucleotide sequence; and iii) a right transposase recognition sequence comprising a sequence having at least 80% sequence identity with SEQ ID NO: 79.

[0040] In some embodiments, the system further includes a PAM sequence compatible with the Cas effector complex. In some embodiments, the PAM sequence includes SEQ ID NO: 31.

[0041] In some embodiments, the PAM sequence is located at approximately 50 to approximately 70 base pairs from the target nucleic acid site. In some embodiments, the PAM sequence is located at the 3' position of the target nucleic acid site. In some embodiments, the PAM sequence is located at the 5' position of the target nucleic acid site.

[0042] In some embodiments, the Cas effector complex further comprises a small prokaryotic ribosomal protein subunit S15. In some embodiments, the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity with any of SEQ ID NO: 187-189. In some embodiments, the accessory protein is ClpX, which comprises a sequence having at least 80% sequence identity with any of SEQ ID NO: 235-249.

[0043] In some aspects, this disclosure provides an engineered nuclease system comprising: a nuclease including a RuvC domain, said nuclease being derived from an uncultured microorganism and being a type 2 VK Cas effector, said type 2 VK Cas effector having at least 80% identity with any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85 and 220; and an engineered guide RNA forming a complex with said nuclease and including a spacer sequence, said spacer sequence hybridizing with a target nucleic acid sequence, wherein said engineered guide polynucleotide includes a sequence having at least 80% identity with any one of SEQ ID NO: 754-944.

[0044] In some aspects, this disclosure provides a method for transposing a cargo nucleotide sequence into a target nucleic acid site, the method comprising introducing any of the systems disclosed herein into a cell. In some aspects, this disclosure provides a cell comprising any of the systems disclosed herein. In some embodiments, the cell is a eukaryotic cell.

[0045] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof. In some embodiments, the cell is an engineered cell. In some embodiments, the cell is a stable cell.

[0046] Further aspects and advantages of this disclosure will become apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of this disclosure are shown and described. It will be appreciated that this disclosure can have other and different embodiments, and that certain details thereof can be modified in various obvious ways without departing from this disclosure. Therefore, the drawings and description are to be regarded in an illustrative rather than restrictive manner. Attached Figure Description

[0047] The novel features of this disclosure are set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description of illustrative embodiments that utilize the principles of this disclosure, and in the accompanying drawings (also referred to herein as “Figure / FIG.”): Figure 1 Example tissues of different kinds and types of CRISPR / Cas loci are depicted.

[0048] Figure 2 The architecture for natural type II crRNA / tracrRNA pairs, such as those shown in Cas9, is depicted in comparison to hybrid sgRNAs in which crRNA and tracrRNA are linked.

[0049] Figure 3 Two pathways found in Tn7 and Tn7-like elements are described.

[0050] Figures 4A-4B The genomic background of V-type Tn7 CAST of the MG64 family was depicted. Figure 4A The MG64-1 CAST system was depicted, comprising a CRISPR array (CRISPR repeat sequences), a type V nuclease, and three predicted transposase protein sequences. tracrRNA was predicted in the intergenic region between the CAST effector and the CRISPR array. The figure below shows multiple sequence alignments of the catalytic domain of transposase TnsB. Catalytic residues are indicated by boxes. Figure 4B The two transposable ends of the predicted MG64-1 CAST system are depicted.

[0051] Figure 5 The predicted structure of the corresponding sgRNA of the CAST system described in this paper is depicted. Figure 5 Figure A (left) shows the predicted MG64-1 tracrRNA and crRNA duplex complex at the repeat-anti-repeat stem. The loop was truncated and a quadruplex of GAAA was added to the stem-loop structure to produce… Figure 5 The designed sgRNA is shown in small figure B (right).

[0052] Figure 6 The results of transposition reactions targeting a plasmid library consisting of NNNNNNNN at the 5' position of the target spacer sequence are depicted. Reaction #1 indicates the presence of the target library, reaction #2 indicates the presence of the donor fragment in both transposition reactions, and reactions #3-5 indicate sg-specific PCR bands corresponding to the appropriate transposition reactions.

[0053] Figures 7A-7D The results of Sanger sequencing are described. Figure 7AThis shows Sanger sequencing of the donor-target junction on the transposon LE in a transposition reaction closer to the left end (LE) of the PAM. The expected sequence is located at the top of the inset, where the predicted transposition event is 61 bp from the PAM. The top chromatogram shows the sequencing results starting from within the donor fragment. A clear signal can be seen at the right end, extending to the donor / target junction (dashed line). This indicates mixing of the transposition products. The bottom chromatogram of the inset shows sequencing from the target to the donor / target junction. The signal from the left is the clearing signal, extending to the junction. Figure 7B Sanger sequencing of the donor-target junction at the right-hand end (RE) of the transposon in the product of the LE closer to PAM is shown. The expected sequence is located at the top of the small plot, where the predicted transposon event is 61 bp from PAM. The top chromatogram shows the sequencing results starting from within the donor fragment. A clear signal can be seen at the left end, all the way to the donor / target junction (dashed line). Figure 7C This is a close-up of the PAM archive. Figure 7D The SeqLogo analysis of NGS events that are closer to PAM motifs indicates a very strong preference for NGTN in PAM motifs.

[0054] Figure 8 A phylogenetic gene tree of Cas12k effector sequences was constructed. The tree was inferred from multiple sequence alignments of 64 Cas12k sequences recovered here (orange and black branches) and 229 reference Cas12k sequences from public databases (grey branches). Orange branches indicate Cas12k effectors confirmed to associate with CAST transposon components.

[0055] Figure 9 The MG64 family CRISPR repeat sequence alignment is shown. The Cas12k CAST CRISPR repeat sequence contains a conserved motif 5' - GNNGGNNTGAAAG - 3'. In MG64-1, the short anti-repeat (RAR) repeat within the CRISPR repeat sequence motif is aligned with the tracrRNA. The MG64 RAR motif appears to define the start and end of the tracrRNA (5' end: RAR1 (TTTC); 3' end: RAR2 (CCNNC)).

[0056] Figure 10A and Figure 10B The secondary structure predicted by CRISPR repeat sequences + tracrRNA in the folded MG64 system was depicted.

[0057] Figure 11AThe MG64-3 CRISPR locus was depicted. The tracrRNA is encoded upstream of the CRISPR array, while the transposon end is encoded downstream (inner black box). Sequences corresponding to portions of the 3' CRISPR repeat sequence and partial spacers are encoded within the transposon (outer box). Self-matching spacers are encoded outside the transposon end.

[0058] Figure 11B The tracrRNA sequence alignments for the various CASTs provided in this paper are depicted. The tracrRNA sequence alignments reveal conserved regions. Specifically, the sequence “TGCTTTC” located at positions 92–98 (top box) is likely important for the tertiary structure of the sgRNA and for its non-contiguous repeat-anti-repetition pairing with the crRNA. The hairpin “CYCC(n6)GGRG” located at positions 265–278 (bottom box) is likely functionally important, such as by locating downstream sequences that pair with the crRNA.

[0059] Figure 12A The predicted structure of MG64-1 sgRNA was depicted.

[0060] Figure 12B The predicted structure of MG64-3 sgRNA was depicted.

[0061] Figure 12C The predicted structure of MG64-5 sgRNA was depicted.

[0062] Figures 13A-13C PCR data demonstrating MG64-1 activity against sgRNA v2-1 were presented. Effector proteins and their TnsB, TnsC, and TniQ proteins were expressed in an in vitro transcription / translation system using the described in vitro targeted integrase activity protocol. After translation, target DNA, cargo DNA, and sgRNA were added to a reaction buffer. Integration was determined by PCR across the target / donor junction. Figure 13A A diagram depicting the possible orientations of integrated donor DNA is shown. PCR reactions 3, 4, 5, and 6 represent each integration ligation product, depending on the orientation of the donor integration at the target site. Figure 13B Gel images depicting transposable PCR 4 (detection of RE binding sites with donor) show: lane 1) apo (without sgRNA); lane 2) with sgRNA 1; and lane 3) with sgRNA v2-1. Figure 13C Gel images depicting transposable PCR 5 (detection of LE binding sites with donor) show: lane 1) apo (without sgRNA); lane 2) with sgRNA 1; and lane 3) with sgRNA v2-1.

[0063] Figure 14PCR reaction 5 (LE near PAM, top half of the figure) and PCR reaction 4 (RE far from PAM, bottom half of the figure) are plotted on the MG64-1 sequence at a certain distance from PAM. Analysis of the integration window shows that 95% of the integration occurring at the spacer PAM site is within a 10 bp window between 58 and 68 nucleotides from PAM. The difference in integration distance between distal and proximal frequencies reflects that the 3-5 base pair repeats at the integration site are a result of the alternating nuclease activity of transposases during integration.

[0064] Figure 15 The results of colony PCR screening for transposition efficiency are depicted. After incubation, 18 colony-forming units (CFUs) were visible on the plates; 8 were on plate A (without IPTG, lane labeled A), and 10 were on plate B (with 100 µM IPTG recovered, lane labeled B). All 18 were analyzed by colony PCR, which shows the product bands (arrows) indicating successful transposition reactions.

[0065] Figure 16 The sequencing results of the selected colony PCR products were depicted, confirming that they represent a transposition event, as they are located in... lacZ The engineered target site in the gene spans the junction between LE and PAM. The minimum LE sequence is shown in blue at the top of the screen (min LE), while the target and PAM are shown in gray. Some sequence variations were observed in the PCR product, but these are expected considering that the insertion may occur at a variable distance upstream of PAM.

[0066] Figure 17 The test results for the 64-1 transposable activity of the engineered unidirectional guide are depicted. The black boxes indicate lanes unrelated to this experiment. Figure 17 Small figure A depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): lane 1 = apo (no sgRNA); lane 2 = holo (+ sgRNA); lane 3 = sgRNA v1-1; lane 4 = sgRNA v1-2; lane 5 = sgRNA v1-3. Figure 17 Small figure B depicts a gel image of transposable PCR 5 (detection of LE binding sites with donor): lane 1 = apo (no sgRNA); lane 2 = holo (+ sgRNA); lane 3 = sgRNA v1-1; lane 4 = sgRNA v1-2; lane 5 = sgRNA v1-3. Figure 17Small figure C depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): lane 1 = apo (no sgRNA); lane 2 = holo (+ sgRNA); lane 3 = sgRNA v1-4; lane 4 = sgRNA v1-6; lane 5 = sgRNA v1-7; lane 6 = sgRNA v1-8; lane 7 = sgRNA v1-9. Figure 17 Small figure D depicts a gel image of transposable PCR 5 (detection of LE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = sgRNA v1-4; Lane 4 = sgRNA v1-6; Lane 5 = sgRNA v1-7; Lane 6 = sgRNA v1-8; Lane 7 = sgRNA v1-9. Figure 17 Small figure E depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): lane 1 = apo (no sgRNA); lane 2 = holo (+ sgRNA); lane 3 = sgRNA v1-5; lane 4 = skipped; lane 5 = sgRNA v1-10. Figure 17 Small figure F depicts a gel image of transposable PCR 5 (detection of LE binding sites with donors): lane 1 = apo (no sgRNA); lane 2 = holo (+ sgRNA); lane 3 = sgRNA v1-5; lane 4 = skipped; lane 5 = sgRNA v1-10. Figure 17 Small figure G depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = sgRNA v1-17; Lane 4 = sgRNA v1-18; Lane 5 = skipped; Lane 6 = sgRNA v1-19; Lane 7 = skipped; Lane 8 = sgRNA v1-20. Figure 17 The small figure H depicts a gel image of transposable PCR 5 (detection of LE binding sites with the donor): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = sgRNA v1-17; Lane 4 = sgRNA v1-18; Lane 5 = skipped; Lane 6 = sgRNA v1-19; Lane 7 = skipped; Lane 8 = sgRNA v1-20.

[0067] Figure 18 The test results for the 64-1 transposable activity of engineered LE and RE are depicted. The black boxes indicate swimlanes unrelated to this experiment. Figure 18 In Figure A, a gel image depicting transposable PCR 4 (detection of RE binding sites with donors) is shown: Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = LE 86 bp; Lane 4 = LE 105 bp; Lane 5 = RE 196 bp; Lane 6 = RE 242 bp; Lane 7 = RE internal deletion 50; Lane 8 = RE internal deletion 81. Figure 18 Small figure B depicts a gel image of transposable PCR 5 (detecting the LE binding site with the donor): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = LE 86 bp; Lane 4 = LE 105 bp; Lane 5 = RE 196 bp; Lane 6 = RE 242 bp; Lane 7 = RE internal deletion 50; Lane 8 = RE internal deletion 81. Figure 18 In small figure C, a gel image of transposable PCR 4 (detection of RE binding sites with donors) is shown: Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = RE internal deletion of 81 and 178 bp; Lane 4 = skipped; Lane 5 = RE internal deletion of 81 and 196 bp; Lane 6 = skipped; Lane 7 = RE internal deletion of 81 and 212 bp; Lane 8 = skipped. Figure 18 Small figure D depicts a gel image of transposable PCR 5 (detection of LE binding sites with donor): Lane 1 = apo (no sgRNA); Lane 2 = holo (+sgRNA); Lane 3 = RE internal deletion of 81 and 178 bp; Lane 4 = skipped; Lane 5 = RE internal deletion of 81 and 196 bp; Lane 6 = skipped; Lane 7 = RE internal deletion of 81 and 212 bp; Lane 8 = skipped. Figure 18 Figure E depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+sgRNA); Lane 3 = RE internal deletion 81 and 178 bp + LE 68 bp; Lane 4 = RE internal deletion 81 and 178 bp + LE 86 bp; Lane 5 = Skipped; Lane 6 = RE internal deletion 81 and 178 bp + LE 105 bp; Lane 7 = Skipped. Figure 18In Figure F, a gel image depicting PCR 5 (detection of LE binding sites with the donor) of transposable ... Figure 18 Small figure G depicts a gel image of PCR 6 (detection of RE binding sites with donor) with transposable ...

[0068] Figure 19 The results of testing the transposable activity of engineered CAST components with NLS are depicted. The black boxes indicate lanes unrelated to this experiment. Figure 19 Figure A depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = skipped; Lane 4 = skipped; Lane 5 = skipped; Lane 6 = NLS-TnsB; Lane 7 = skipped; Lane 8 = TnsB-NLS. Figure 19 Small figure A depicts a gel image of transposable PCR 5 (detection of LE binding sites with donor): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = skipped; Lane 4 = skipped; Lane 5 = skipped; Lane 6 = NLS-TnsB; Lane 7 = skipped; Lane 8 = TnsB-NLS. Figure 19 Small figure C depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = skip; Lane 4 = skip; Lane 5 = skip; Lane 6 = NLS-TniQ; Lane 7 = skip; Lane 8 = TniQ-NLS. Figure 19The small figure D depicts a gel image of transposable PCR 5 (detection of LE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = skipped; Lane 4 = skipped; Lane 5 = skipped; Lane 6 = NLS-TniQ; Lane 7 = skipped; Lane 8 = TniQ-NLS. Figure 19 Figure E depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = skipped; Lane 4 = skipped; Lane 5 = NLS-Cas12k; Lane 6 = Cas12k-NLS; Lane 7 = NLS-TnsC; Lane 8 = TnsC-NLS. Figure 19 Figure F depicts a gel image of transposable PCR 5 (detection of LE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = skipped; Lane 4 = skipped; Lane 5 = NLS-Cas12k; Lane 6 = Cas12k-NLS; Lane 7 = NLS-TnsC; Lane 8 = TnsC-NLS. Figure 19 Small figure G depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = NLS-HA-TnsC; Lane 4 = NLS-TnsC-FLAG; Lane 5 = NLS-TnsC-HA; Lane 6 = NLS-TnsC-Myc; Lane 7 = NLS-FLAG-TnsC; Lane 8 = NLS-Myc-TnsC. Figure 19 The small figure H depicts a gel image of transposable PCR 5 (detecting the LE binding site with the donor): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = NLS-HA-TnsC; Lane 4 = NLS-TnsC-FLAG; Lane 5 = NLS-TnsC-HA; Lane 6 = NLS-TnsC-Myc; Lane 7 = NLS-FLAG-TnsC; Lane 8 = NLS-Myc-TnsC. Figure 19Figure I depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = Cas 2x NLS apo (no sgRNA); Lane 4 = Cas 2x NLS holo (+ sgRNA). Figure 19 Figure J depicts a gel image of transposable PCR 5 (detection of LE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = Cas 2x NLS apo (no sgRNA); Lane 4 = Cas 2x NLS holo (+ sgRNA).

[0069] Figure 20 The engineered CAST-NLS is depicted as a single kit. Unless otherwise described, all lanes contain Cas12k-NLS and NLS-TniQ, TnsB, TnsC, and sgRNA. Figure 20 Small figure A depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = NLS-TnsB; Lane 4 = TnsB-NLS; Lane 5 = NLS-TnsB and NLS-TnsC; Lane 6 = TnsB-NLS and NLS-TnsC. Figure 20 Small figure B depicts a gel image of transposable PCR 5 (detection of LE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = NLS-TnsB; Lane 4 = TnsB-NLS; Lane 5 = NLS-TnsB and NLS-TnsC; Lane 6 = TnsB-NLS and NLS-TnsC.

[0070] Figure 21 The results of the transposition activity assays of the Cas effector and the TniQ protein fusion were described. Figure 21 Small figure B depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (without sgRNA) fusion with Cas-TniQ; Lane 2 = holo (+ sgRNA) fusion with Cas-TniQ; Lane 3 = apo (without sgRNA) fusion with TniQ-Cas; Lane 4 = holo (+ sgRNA) fusion with TniQ-Cas. Figure 21Small figure B depicts a gel image of transposon PCR 5 (detection of LE binding sites with donors): Lane 1 = apo (without sgRNA) fusion with Cas-TniQ; Lane 2 = holo (+ sgRNA) fusion with Cas-TniQ; Lane 3 = apo (without sgRNA) fusion with TniQ-Cas; Lane 4 = holo (+ sgRNA) fusion with TniQ-Cas. Figure 21 Small figure C depicts a gel image of transposon PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (without sgRNA) fusion with TniQ-Cas; Lane 2 = holo (+ sgRNA) fusion with TniQ-Cas; Lane 3 = holo Cas alone; Lane 4 = apo (without sgRNA) fusion with TniQ-48 adapter-Cas; Lane 5 = holo (+ sgRNA) fusion with TniQ-48 adapter-Cas; Lane 6 = apo (without sgRNA) fusion with TniQ-68 adapter-Cas; Lane 7 = holo (+ sgRNA) fusion with TniQ-68 adapter-Cas; Lane 8 = holo (+ sgRNA) fusion with TniQ-72 adapter-Cas. Figure 21 Small figure D depicts a gel image of transposon PCR 5 (detection of LE binding sites with donors): Lane 1 = apo (without sgRNA) fusion with TniQ-Cas; Lane 2 = holo (+ sgRNA) fusion with TniQ-Cas; Lane 3 = holo Cas alone; Lane 4 = apo (without sgRNA) fusion with TniQ-48 linker-Cas; Lane 5 = holo (+ sgRNA) fusion with TniQ-48 linker-Cas; Lane 6 = apo (without sgRNA) fusion with TniQ-68 linker-Cas; Lane 7 = holo (+ sgRNA) fusion with TniQ-68 linker-Cas; Lane 8 = holo (+ sgRNA) fusion with TniQ-72 linker-Cas. Figure 21Figure E depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (without sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = apo (without sgRNA) with NLS-TniQ-Cas-NLS fusion; Lane 4 = holo (+ sgRNA) with NLS-TniQ-Cas-NLS fusion; Lane 5 = apo (without sgRNA) with NLS-TniQ-77 adapter-Cas-NLS fusion; Lane 6 = holo (+ sgRNA) with NLS-TniQ-77 adapter-Cas-NLS fusion. Figure 21 Figure F depicts a gel image of transposable PCR 5 (detection of LE binding sites with donors): Lane 1 = apo (without sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = apo (without sgRNA) with NLS-TniQ-Cas-NLS fusion; Lane 4 = holo (+ sgRNA) with NLS-TniQ-Cas-NLS fusion; Lane 5 = apo (without sgRNA) with NLS-TniQ-77 adapter-Cas-NLS fusion; Lane 6 = holo (+ sgRNA) with NLS-TniQ-77 adapter-Cas-NLS fusion. Figure 21 Small figure G depicts the gel image of transposon PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = NLS-TniQ-Cas-NLS apo (no sgRNA); Lane 4 = NLS-TniQ-Cas-NLS holo (+ sgRNA); Lane 5 = Cas-NLS-P2A-NLS-TniQapo (no sgRNA); Lane 6 = Cas-NLS-P2A-NLS-TniQ holo (+ sgRNA). Figure 21 The small figure H depicts the gel image of transposon PCR 5 (detection of LE binding site with donor): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = NLS-TniQ-Cas-NLS apo (no sgRNA); Lane 4 = NLS-TniQ-Cas-NLS holo (+ sgRNA); Lane 5 = Cas-NLS-P2A-NLS-TniQ apo (no sgRNA); Lane 6 = Cas-NLS-P2A-NLS-TniQholo (+ sgRNA).

[0071] Figure 22The expression of TnsB and TnsC in human cells was depicted, followed by cell grading and results of in vitro transposition reactions. Figure 22 Small figure A depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (without sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = holo (+ sgRNA) with untreated (TnsB-free) cytoplasm; Lane 4 = holo (+ sgRNA) with untreated nucleoplasm; Lane 5 = holo (+ sgRNA) with NLS-TnsB cell cytoplasm; Lane 6 = holo (+ sgRNA) with NLS-TnsB cell nucleoplasm; Lane 7 = holo (+ sgRNA) with TnsB-NLS cell cytoplasm; Lane 8 = holo (+ sgRNA) with TnsB-NLS cell nucleoplasm; Lane 9 = holo (+ sgRNA) with NLS-TniQ cell cytoplasm; Lane 10 = holo (+ sgRNA) with NLS-TniQ cell nucleoplasm. Figure 22 Small figure B depicts a gel image of transposable PCR 5 (detection of LE binding sites with donors): Lane 1 = apo (without sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = holo (+ sgRNA) with untreated (TnsB-free) cytoplasm; Lane 4 = holo (+ sgRNA) with untreated nucleoplasm; Lane 5 = holo (+ sgRNA) with NLS-TnsB cell cytoplasm; Lane 6 = holo (+ sgRNA) with NLS-TnsB cell nucleoplasm; Lane 7 = holo (+ sgRNA) with TnsB-NLS cell cytoplasm; Lane 8 = holo (+ sgRNA) with TnsB-NLS cell nucleoplasm; Lane 9 = holo (+ sgRNA) with NLS-TniQ cell cytoplasm; Lane 10 = holo (+ sgRNA) with NLS-TniQ cell nucleoplasm. Figure 22Small figure C depicts a gel image of transposable PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (without sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = holo (+ sgRNA) without TnsC; Lane 4 = holo (+ sgRNA) with untreated (TnsC-free) cytoplasm; Lane 5 = holo (+ sgRNA) with untreated nucleoplasm; Lane 6 = holo (+ sgRNA) with NLS-HA-TnsC cell cytoplasm; Lane 7 = holo (+ sgRNA) with NLS-HA-TnsC cell nucleoplasm; Lane 8 = holo (+ sgRNA) with TnsC-NLS cell cytoplasm; Lane 9 = holo (+ sgRNA) with TnsC-NLS cell nucleoplasm. Figure 22 Small figure D depicts a gel image of transposable PCR 5 (detection of LE binding sites with donors): Lane 1 = apo (without sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = holo (+ sgRNA) without TnsC; Lane 4 = holo (+ sgRNA) with untreated (TnsC-free) cytoplasm; Lane 5 = holo (+ sgRNA) with untreated nucleoplasm; Lane 6 = holo (+ sgRNA) with NLS-HA-TnsC cell cytoplasm; Lane 7 = holo (+ sgRNA) with NLS-HA-TnsC cell nucleoplasm; Lane 8 = holo (+ sgRNA) with TnsC-NLS cell cytoplasm; Lane 9 = holo (+ sgRNA) with TnsC-NLS cell nucleoplasm. Figure 22Small figure E depicts the gel image of PCR 4 (detection of RE binding sites with donors) for transposition: Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = apo (no sgRNA) NLS-TnsB-IRES-NLS-TnsC cytoplasm; Lane 4 = holo (+ sgRNA) NLS-TnsB-IRES-NLS-TnsC cytoplasm; Lane 5 = apo (no sgRNA) NLS-TnsB-IRES-NLS-TnsC nucleoplasm; Lane 6 = holo (+ sgRNA) NLS-TnsB-IRES-NLS-TnsC nucleoplasm; Lane 7 = apo (no sgRNA) TnsB-NLS-IRES-NLS-TnsC cytoplasm; Lane 8 = holo (+ sgRNA) TnsB-NLS-IRES-NLS-TnsC cytoplasm; Lane 9 = apo (without sgRNA) TnsB-NLS-IRES-NLS-TnsC nucleoplasm; Lane 10 = holo (+sgRNA) TnsB-NLS-IRES-NLS-TnsC nucleoplasm. Figure 22 Small figure F depicts the gel image of PCR 5 (detection of LE binding sites with donor) for transposation: Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = apo (no sgRNA) NLS-TnsB-IRES-NLS-TnsC cytoplasm; Lane 4 = holo (+ sgRNA) NLS-TnsB-IRES-NLS-TnsC cytoplasm; Lane 5 = apo (no sgRNA) NLS-TnsB-IRES-NLS-TnsC nucleoplasm; Lane 6 = holo (+ sgRNA) NLS-TnsB-IRES-NLS-TnsC nucleoplasm; Lane 7 = apo (no sgRNA) TnsB-NLS-IRES-NLS-TnsC cytoplasm; Lane 8 = holo (+ sgRNA) TnsB-NLS-IRES-NLS-TnsC cytoplasm; Lane 9 = apo (without sgRNA) TnsB-NLS-IRES-NLS-TnsC nucleoplasm; Lane 10 = holo (+ sgRNA) TnsB-NLS-IRES-NLS-TnsC nucleoplasm.

[0072] Figure 23 The expression of the Cas12k and TniQ linked construct in human cells was depicted, followed by the results of in vitro transposition assays. Figure 23Small figure A depicts a gel image of transposable PCR 5 (detection of LE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = Cas-NLS holo (+ sgRNA) cytoplasm; Lane 4 = Cas-NLS holo (+ sgRNA) nucleoplasm; Lane 5 = Cas-NLS holo (+ sgRNA) nucleoplasm + additional sgRNA; Lane 6 = Cas-NLS-P2A-NLS-TniQ holo (+ sgRNA) cytoplasm; Lane 7 = Cas-NLS-P2A-NLS-TniQ holo (+ sgRNA) nucleoplasm; Lane 8 = Cas-NLS-P2A-NLS-TniQ holo (+ sgRNA) nucleoplasm + additional sgRNA. Figure 23 Small figure B depicts the gel image of transposable PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = apo (no sgRNA) Cas-NLS-P2A-NLS-TniQ cytoplasm; Lane 4 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ cytoplasm; Lane 5 = apo (no sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm; Lane 6 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm; Lane 7 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm + additional holo Cas-NLS; Lane 8 ...9 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm + additional holo Cas-NLS; Lane 10 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm + additional holo Cas-NLS; Lane 11 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm + additional holo Cas-NLS nucleoplasm; Lane 12 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm + additional holo Cas-NLS nucleop sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasmic+NLS-TniQ. Figure 23Small figure C depicts the gel image of PCR 5 (detection of LE binding sites with donors) with transposable transposons: Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = apo (no sgRNA) Cas-NLS-P2A-NLS-TniQ cytoplasm; Lane 4 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ cytoplasm; Lane 5 = apo (no sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm; Lane 6 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm; Lane 7 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm + additional holo Cas-NLS; Lane 8 ...9 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm + additional holo Cas-NLS; Lane 10 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm + additional holo Cas-NLS; Lane 11 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm + additional holo Cas-NLS nucleoplasm; Lane 12 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm + additional holo Cas sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasmic+NLS-TniQ. Figure 23 Small figure D depicts the gel image of transposable PCR 4 (detection of RE binding sites with donors): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = apo (no sgRNA) NLS-TniQ-Cas-NLS cytoplasm; Lane 4 = holo (+ sgRNA) NLS-TniQ-Cas-NLS cytoplasm; Lane 5 = apo (no sgRNA) NLS-TniQ-Cas-NLS nucleoplasm; Lane 6 = holo (+ sgRNA) NLS-TniQ-Cas-NLS nucleoplasm; Lane 7 = holo (+ sgRNA) NLS-TniQ-Cas-NLS nucleoplasm + additional holo Cas-NLS; Lane 8 = holo (+ sgRNA) NLS-TniQ-Cas-NLS nucleoplasm + NLS-TniQ. Figure 23Small figure E depicts the gel image of transposable PCR 5 (detection of LE binding sites with donors): Lane 1 = apo (without sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = apo (without sgRNA) NLS-TniQ-Cas-NLS cytoplasm; Lane 4 = holo (+ sgRNA) NLS-TniQ-Cas-NLS cytoplasm; Lane 5 = apo (without sgRNA) NLS-TniQ-Cas-NLS nucleoplasm; Lane 6 = holo (+ sgRNA) NLS-TniQ-Cas-NLS nucleoplasm; Lane 7 = holo (+ sgRNA) NLS-TniQ-Cas-NLS nucleoplasm + additional holo Cas-NLS; Lane 8 = holo (+ sgRNA) NLS-TniQ-Cas-NLS nucleoplasm + NLS-TniQ. Figure 23 Small figure F depicts the gel image of PCR 4 (detection of RE binding sites with donors) with transposition: Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ cytoplasm; Lane 4 = holo (+ sgRNA) Cas-NLS-IRES-NLS-TniQ cytoplasm; Lane 5 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasm; Lane 6 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasm + additional PURExpress; Lane 7 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasm + additional Cas-NLS; Lane 8 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasm + NLS-TniQ; Lane 9 = holo(+ sgRNA)Cas-NLS-IRES-NLS-TniQ nucleoplasm; Lane 10 = holo(+ sgRNA)Cas-NLS-IRES-NLS-TniQ nucleoplasm + additional PURExpress; Lane 11 = holo(+ sgRNA)Cas-NLS-IRES-NLS-TniQ nucleoplasm + additional Cas-NLS; Lane 12 = holo(+ sgRNA)Cas-NLS-IRES-NLS-TniQ nucleoplasm + NLS-TniQ. Figure 23Small figure G depicts the gel image of PCR 5 (detection of LE binding sites with donor) for transposation: Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ cytoplasm; Lane 4 = holo (+ sgRNA) Cas-NLS-IRES-NLS-TniQ cytoplasm; Lane 5 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasm; Lane 6 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasm + additional PURExpress; Lane 7 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasm + additional Cas-NLS; Lane 8 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasm + NLS-TniQ; Lane 9 = holo(+ sgRNA)Cas-NLS-IRES-NLS-TniQ nucleoplasm; Lane 10 = holo(+ sgRNA)Cas-NLS-IRES-NLS-TniQ nucleoplasm + additional PURExpress; Lane 11 = holo(+ sgRNA)Cas-NLS-IRES-NLS-TniQ nucleoplasm + additional Cas-NLS; Lane 12 = holo(+ sgRNA)Cas-NLS-IRES-NLS-TniQ nucleoplasm + NLS-TniQ.

[0073] Figure 24 Electrophoretic mobility shift assay (EMSA) results for 64-1 TnsB and its LE DNA sequence were depicted. EMSA results confirmed binding and TnsB recognition. The TnsB protein was expressed in an in vitro transcription / translation system, incubated with FAM-tagged DNA containing the LE sequence, and then separated on a natural 5% TBE gel. Binding was observed as an upward shift in the labeled band. Multiple TnsB binding sites resulted in multiple shifts in the EMSA. Lane 1: FAM-tagged DNA only. Lane 2: FAM DNA plus in vitro transcription / translation system (without TnsB protein). Lane 3: FAM DNA plus TnsB.

[0074] Figures 25A-25B The diversity of Cas12k effectors was described. Figure 25AThe Cas12k CAST genome background is depicted. Transposons are characterized by: terminal inverted repeat sequences (TIR, light orange bar), Tn7-like transposon genes (colored arrows), the death effector Cas12k (orange arrow), tracrRNA (pink half-arrow), and a CRISPR array. The “TAAA” target site repeat (TDS) is observed flanking the TIR. Middle inset: Inset of the MG64-1 untranslated region, showing tracrRNA (a pseudo-repetitive sequence and self-targeting spacer), the CRISPR array, and the left-hand TIR of the transposon. Bottom inset: Multiple alignment of pseudo-repetitive sequences and self-targeting spacers in a set of CAST homologs. Figure 25B A rootless phylogenetic tree of Cas12k effectors was constructed. Cas12k effectors recovered in this study are shown as orange (transposons already identified in the genome) and black branches, while reference Cas12k sequences are shown in gray. Reference sequences ShCas12k and AcCas12k are indicated by red arrows.

[0075] Figures 26A-26B The right end of CAST was depicted ( Figure 26A ) and the left end ( Figure 26B Multiple sequence alignment. The transposon inverted motif “TGTNNA” is highlighted with a box.

[0076] Figure 27 The alignment of the Cas12k CAST tracrRNA sequence was depicted, revealing regions of sequence and structural conservation. Specifically, the sequence “TGCTTTC” located at positions 88–92 is likely important for the tertiary structure of the sgRNA and for its discontinuous repeat-anti-repetition pairing with the crRNA. The hairpin “CYCC(n6)GGRG” located at positions 279–294 is likely functionally important and may locate the downstream sequence for crRNA pairing.

[0077] Figure 28 The single-guide RNA folding of the active MG64-1, MG64-2, and MG64-6 CAST systems was depicted. The active engineered sgRNA of MG64-1 was also shown.

[0078] Figures 29A-29B In vitro screening of CAST transposons using PAM libraries is described. Figure 29A The screening settings determined by in vitro PAM are described. Figure 29B A schematic diagram of junctional PCR used to detect transposon products is depicted.

[0079] Figure 30A The transposon junctions of MG64-1 CAST (left lane) and MG64-6 CAST (right lane) amplified by PCR were depicted.

[0080] Figure 30B The SeqLogo representation (top) depicts the PAM detected by MG64-1.

[0081] Figure 30C The integrated frequencies plotted by distance at the proximal and distal distances of MG64-1 were depicted.

[0082] Figure 31 The engineering of the 64-1 single-guide RNA was depicted. The deletion of the region from approximately 130 bp to 190 bp (the green and cyan segments of the structure) produces an sgRNA that guides a strong transposition reaction (green bars on the heatmap).

[0083] Figure 32 The cross-reactivity of MG64-2 sgRNA with MG64-1 and PAM was described for the combination of MG64-2 sgRNA and MG64-1 effector.

[0084] Figure 33 Single guide RNA truncations in the encoding DNA were depicted for MG64-2 sgRNA in both the sequence view and the secondary structure prediction model. Missing regions and truncations in the sequence view are shown as bars (del1, del2, del3, del4, del5, and del6). Missing regions in the ellipses in the secondary structure prediction model indicate the truncations tested (pseudoknots, deletions 1, 2, 3, and 4 on 5 and 6).

[0085] Figure 34 Data demonstrating the activity of engineered MG64-2 sgRNA against the MG64-1 CAST system are presented. PCR reactions are shown for each possible integration site or negative control (small plot B in Figure 29). Successful integration products are highlighted with arrows. Boxed lanes are irrelevant to this experiment.

[0086] Figure 35 The design of the MG64-2 sgRNA splitting guide was described. sgRNA fragments were synthesized separately and then re-annealed before testing in transposition experiments.

[0087] Figure 36 Data demonstrating the activity of the cleaved MG64-2 sgRNA against the MG64-1 CAST system are presented. PCR reactions are shown for each possible integration site or negative control (small plot B in Figure 29). Successful integration products are highlighted with arrows. Boxed lanes are irrelevant to this experiment.

[0088] Figure 37 Data demonstrating that minimizing LE and RE maintains the transposability activity of the system are presented.

[0089] Figures 38A-38CDescribes Escherichia coli ( E. coli The result of integrating with MG64-1. Figure 38A A schematic representation of the introduction of the CAST system into Escherichia coli is depicted. Figure 38B The NGS data depicts an editing efficiency greater than 80%. Figure 38C An off-target analysis was depicted, showing that no off-target integration greater than 1% of all summed transposition events was detected.

[0090] Figure 39 The local insertion rates of various endogenous loci in the Escherichia coli genome were depicted.

[0091] Figures 40A-40B The results of multi-locus targeting were described. Figure 40A The corresponding local insertion frequencies at endogenous and engineered loci were depicted. Figure 40B The relative insertion frequencies of targeted insertions at endogenous loci, targeted insertions at engineered loci, and off-target insertions were depicted. Integrations at the two loci combined account for more than 95% of all integrations occurring on the genome.

[0092] Figure 41 Sanger sequencing data depicting the integrated PCR product demonstrates the in vitro activity of MG64-1. The reaction is a RE donor-target product reaction, and sequencing stops when the junction appears to match the donor DNA (dark bar below the sequencing peak).

[0093] Figure 42A A schematic representation of serially diluted target DNA for in vitro transposition experiments is shown. The CAST component was expressed and added to a reaction solution containing in vitro transcribed sgRNA and a donor plasmid. Target plasmid DNA was added at a reduced concentration, and transposition experiments were performed. Once the minimum amount of target DNA was determined, the transposition reaction was measured by adding an increased amount of human genomic DNA.

[0094] Figure 42B A diagram illustrating PCR amplification of the transposable reaction is shown. An 8N PAM plasmid library (8N-Target, Rxn #1) was targeted using the CAST system to integrate donor DNA (Rxn #2). After successful integration, conjugate site PCR was performed with primers to amplify four putative integration reactions (Rxn #3, #4, #5, and #6) based on the orientation of cargo integration.

[0095] Figure 42C The PCR reaction products from an in vitro transposition assay performed using serially diluted target plasmid DNA are shown. Target, donor, and reactions #3, #4, #5, and #6 correspond to... Figure 42B The PCR integration product shown.

[0096] Figure 42D The PCR reaction products from an in vitro transposition assay using a fixed amount of target plasmid DNA (0.5 ng) are shown, with the addition of an increased amount of human genomic DNA to expand the search space. Target, donor, and reactions #3, #4, #5, and #6 correspond to... Figure 42B The PCR integration product shown.

[0097] Figure 43A A schematic diagram of a transposition reaction across a high-copy element is shown. When measured with CAST protein and sgRNA targeting one of multiple aligned targets, the target PCR product spans the wild-type target element. Integration can occur in forward orientation, reverse orientation, or both. Forward transposition products are measured by conjugate PCR amplifying the region covering the LE of the donor DNA to the 5' end of the target site (forward PCR). Reverse conjugate reaction measures the region covering the LE of the donor DNA to the 3' end of the target element (reverse PCR).

[0098] Figure 43B The PCR reaction products from in vitro transposition assays are shown at 15 target sites (guides) of the LINE1 3' element in human genomic DNA. Targets and reactions: Forward and reverse PCR correspond as follows: Figure 43A The PCR integration product shown.

[0099] Figure 43C This diagram shows PCR reaction products from in vitro transposition assays at 15 target sites (guides) of SVA elements in human genomic DNA. Targets and reactions are shown for forward and reverse PCR, as follows: Figure 43A The PCR integration product is shown. The bands are highlighted with arrows indicating successful targeted integration.

[0100] Figure 43D This diagram shows PCR reaction products from in vitro transposition assays at 15 target sites (guides) of HERV elements in human genomic DNA. Targets and reactions are shown for forward and reverse PCR, as follows: Figure 43A The PCR integration product is shown. The bands are highlighted with arrows indicating successful targeted integration.

[0101] Figure 43E Sanger sequencing of the forward PCR integration products at multiple target sites on the LINE1 3' element is shown. At the sites of integration, the sequencing trace stops matching the donor DNA (gray vertical bars).

[0102] Figure 43FSanger sequencing of the inverse PCR integration products at multiple target sites on the LINE1 3' element is shown. At the sites of integration, the sequencing trace stops matching the target DNA (gray vertical bars).

[0103] Figure 43G Sanger sequencing of the forward PCR integration product at SVA target site 3 is shown. At the site of integration, the sequencing trace stops matching the donor DNA (gray vertical bar).

[0104] Figure 43H Sanger sequencing of the forward PCR integration product at HERV target site 5 is shown. At the site of integration, the sequencing trace stops matching the donor DNA (gray vertical bar).

[0105] Figure 44 The PCR reaction products from in vitro transposition assays are shown at LINE1 target sites 12 and 15 in human genomic DNA containing functional domains. Target and reaction: Forward and reverse PCR correspond as follows: Figure 42A The PCR integration product is shown. The bands are highlighted with arrows indicating successful targeted integration.

[0106] Figures 45A-45B In vitro transposition experiments using CAST, S15, NLS-S15, and S15-NLS expressed by eukaryotic transcription / translation responses are demonstrated. Figure 45A The in vitro transposition reaction using MG64-1 CAST and S15 is shown. The CAST component expressed by wheat germ extract promotes transposition without the addition of S15, although at a low rate (weak band highlighted by arrows). The addition of PURExpress reagent (used PUREx) increases transposition efficiency, as indicated by the intensity of the band at Rxn#5 (PURExpress reagent contains S15). Independent addition of S15 and S15-NLS, which are reactively translated from wheat germ extract, improves the in vitro transposition efficiency of MG64-1 compared to other conditions tested (strong band highlighted by arrows). Figure 45B The in vitro reaction of the transposus with the NLS-S15 configuration is shown. The addition of the PURExpress reagent increased in vitro transposus compared to the CAST component alone (lane 2) (lane 3). The NLS-S15 configuration did not improve transposus (lanes 4-5). Box Rxn#5 indicates the expected band if transposus activity is detected.

[0107] Figures 46A-46H A schematic diagram of a fusion plasmid used for intracellular transposition is shown. Figure 46A Two targeting composite material particles and one donor plasmid were assembled for high-copy elements Line1, targets 8, 12 and 15, and SVA target 3. Figure 46B The diagram illustrates intracellular transposition of high-copy elements at LINE1 targets 8, 12, and 15, and SVA target 3 using H1core-TniQ or HMGN1-TniQ. Arrows indicate transposon junction reactions amplified in the forward (forward PCR) or reverse (reverse PCR) orientation of the transposon. Simulated controls represent reactions without targeting or donor plasmids. Figure 46C Sanger sequencing of the forward PCR of the PCR integration product at target site 8 of LINE1 3' is shown. Integration was mediated by the MG64-1 fusion with the NLS-H1core-TniQ fusion compound. At the site of integration, the sequencing trace stops matching the donor DNA (gray vertical bar). Figure 46D Sanger sequencing of the forward PCR of the integration product at the LINE1 3' target site 8 is shown. Integration was mediated by the MG64-1 fusion with the NLS-HMGN1-TniQ fusion compound. At the site of integration, the sequencing trace stops matching the donor DNA (gray vertical bar). Figure 46E Sanger sequencing of the inverse PCR product of the PCR integration at LINE1 3' target site 12 is shown. Integration was mediated by MG64-1 and the NLS-H1core-TniQ fusion compound. At the site of integration, the sequencing trace stops matching the donor DNA (gray vertical bar). Figure 46F Sanger sequencing of the inverse PCR product of the PCR integration at LINE1 3' target site 12 is shown. Integration was mediated by the MG64-1 fusion with the NLS-HMGN1-TniQ fusion compound. At the site of integration, the sequencing trace stops matching the donor DNA (gray vertical bar). Figure 46G Sanger sequencing of the forward PCR of the PCR integration product at LINE1 3' target site 15 is shown. Integration was mediated by MG64-1 and the NLS-H1core-TniQ fusion compound. At the site of integration, the sequencing trace stops matching the donor DNA (gray vertical bar). Figure 46H Sanger sequencing of the forward PCR of the integration product at LINE1 3' target site 15 is shown. Integration was mediated by the MG64-1 fusion with the NLS-HMGN1-TniQ fusion compound. At the site of integration, the sequencing trace stops matching the donor DNA (gray vertical bar).

[0108] Figures 47A-47D Immunofluorescence staining for locating Cas12k CAST components in human cells was depicted. Figure 47ATop row: Detection of TnsB localization; Middle row: Detection of Cas12k localization; Bottom row: Detection of TnsC localization. Images show that MG64-1 Cas12k and TnsB are localized in the nucleus of mammalian cells, while TnsC is localized in the cytoplasm. Cas12k CAST protein was tagged with HA. Anti-HA antibody was used for protein detection. DAPI was used to stain DNA (nucleus). Figure 47B Top and bottom rows: Detection of TniQ localization. Images show MG64-1 TniQ localized in the nucleus of mammalian cells. CAST protein was tagged with HA. Anti-HA antibody was used for protein detection. DAPI was used to stain DNA (nucleus). Figure 47C All rows: Detection of co-localization of TnsC and TniQ. The image shows that while some TnsC may remain in the cytoplasm, it is now co-localized with TniQ in the nucleus. CAST protein was tagged with HA. Anti-HA antibody was used for protein detection. DAPI was used to stain DNA (nucleus). Figure 47D Two rows: Cas12k, TnsB, TnsC, and TniQ, co-delivered to HEK293T cells, are localized in the nucleus. CAST protein is tagged with HA. Anti-HA antibody is used for protein detection. DAPI is used to stain DNA (nucleus).

[0109] Figures 48A-48B In vitro screening of MG64-1 Cas12k CAST transposons was described. Figure 48A : Graph of the construct used for purification of the MG64-1 holo complex. Figure 48B : Schematic diagram of conjugation PCR used to detect transposon products. The target substrate (target, Rxn#1) with a 5' PAM followed by a protospacer is targeted using the CAST system to integrate cargo DNA (Rxn#2). After successful integration, conjugation PCR is performed with primers to amplify four putative integration reactions based on the cargo integration orientation.

[0110] Figures 48C-48D depict the purification of the MG64-1 protein. Figure 48C: Fractions collected during 2 L-scale purification of the MG64-1 holo complex were run on an unstained denaturing PAGE gel. Figure 48D: Size exclusion chromatography (SEC) chromatogram of the MG64-1 holo complex. The peak centered at 29.3 mL (peak 1) was used for in vitro activity assay.

[0111] Figure 49AThe in vitro transposition of the holo complex, recovered with a TnT-expressing component supplemented in peak 1, is depicted. Lane L) ladder; Lane 1) TnT-expressing CAST component apo condition (-sgRNA); Lane 2) TnT-expressing CAST component holo condition (+sgRNA); Lane 3) Purified peak 1 supplemented with TnT CAST component without additional Cas12k supplementation (-TnTCas12k); Lane 4) Purified peak 1 supplemented with TnT CAST component without additional TnsC supplementation (-TnTTnsC); Lane 5) Peak 1 supplemented with TnT CAST component without additional TniQ supplementation (-TnTTniQ); Lane 6) Peak 1 supplemented with TnT CAST component without additional S15 supplementation (-TnTS15).

[0112] Figure 49B Sanger sequencing of lanes 3, 4, 5, and 6 is depicted from the amplified LE to the pDonor of the PAM target-donor junction and the target orientation. Vertical lines represent the transposon junctions of MG64-1 predicted in the reference sequence. Signal degradation from any direction is caused by the large amount of signal reflected in the PCR amplification.

[0113] Figure 50 Identification of ribosomal protein S15 homologs in cyanobacterial genome fragments was depicted. Candidate sequences from the same sample recovered from MG64-1 are highlighted with solid black circles. (From *E. coli*...) E. coli Reference S15 is indicated by an arrow.

[0114] Figure 51A and 51B Depicting dual transcripts ( Figure 51A ) relative to the integrated transposable component ( Figure 51B A schematic diagram of the two-transcriptional system. On one plasmid, one transcript is controlled by the CMV-βGlobin promoter. Cas12k-sso7d-NLS is linked to S15 via a 2A self-cleaving peptide, and an IRES element separates the second ORF, the NLS-functional domain-TniQ, where the functional domain is either H1-core or HMGN1. The plasmid also contains either a non-targeted (empty) MG64-1 single-guide or a targeted MG64-1 single-guide. On the second plasmid, the CMV-βGlobin promoter drives transcription of NLS-TnsB and NLS-TnsC, separated by an IRES element, and TIRs (LE and RE) are flanked by bacterial origin of replication and antibiotic resistance markers. Under single-transcriptional conditions ( Figure 51BA single CMV-βGlobin promoter controls the expression of the "integrated" transcript, in which Cas12k-sso7d-NLS, S15-NLS, NLS-functional domain-TniQ, NLS-TnsB, and NLS-TnsC are separated by 2A and IRES elements. This single helper plasmid (pHelper) also contains a non-targeted or targeted MG64-1 guide under the control of the pU6 promoter. The second plasmid is pDonor, in which LE and RE are flanked by reporter genes, therapeutic transcripts, or selection markers.

[0115] Figure 52 The test of the integrated pHelper H1core plasmid in human cells is depicted. In the forward LE image, all transposons are shown as conjugate bands. Lane 1: Testing of a dual transcript system with non-targeting single guide ( Figure 51A Lane 2: Test dual transcript system with a targeted single guide encoding for integration into Line 1 3' target 8. Lanes 3-5: Test single transcript pHelper system with pDonor expressing the fluorescent protein mNeon. Lane 3: Integrated pHelper and mNeon pdonor without a targeted single guide. Lane 4: Integrated pHelper and mNeon pDonor with a guide targeting Line 1 3' target 8 at a ratio of 6 µg pHelper to 12 µg pDonor. Lane 5: Integrated pHelper and mNeon pDonor with a guide targeting Line 1 3' target 8 at a ratio of 12 µg pHelper to 6 µg pDonor.

[0116] Figure 53 The test results for non-replicating donors versus replicating donors were depicted. The SV40 origin element of the non-replicating donor was truncated, allowing the plasmid to spread in human cells. Integration was performed using an integrated vector. Lane 1: Simulated transfection control. Lane 2: Integrated pHelper with replicating donor targeting Line 1 3' target 8, as a positive control. Lanes 3-5: Integrated pHelper with replicating mNeon donor without targeting single guide. Lanes 6-8: Integrated pHelper with replicating mNeon donor with Line 1 3' target 8 single guide. Lanes 9-11: Integrated pHelper with non-replicating mNeon donor without targeting single guide. Lanes 12-14: Integrated pHelper with non-replicating mNeon donor with Line 1 3' target 8 single guide.

[0117] Figure 54The addition of ClpX for transposition in cells is depicted. Lane 1: An integrated pHelper with a non-targeting, single-guided mNeon pDonor for transposition. Lane 2: An integrated mNeon donor targeting Line 1 3' target 8, serving as a positive control. Lanes 3-6: Conditions identical to lane 2, but with (0.25 µg, 0.5 µg, 1 µg, and 2 µg) amounts of ClpX-NLS plasmid added to the transposition plasmid mixture. Lanes 7-10: Conditions identical to lane 2, but with (0.25 µg, 0.5 µg, 1 µg, and 2 µg) amounts of NLS-ClpX plasmid added to the transposition plasmid mixture.

[0118] Figure 55 A schematic diagram of the three-plasmid system transfection targeting a single-copy locus is shown. The pHelper plasmid contains a single guide targeting the single-copy locus of interest. A replication plasmid was used for all plasmids, pHelper, pDonor, and pClpX. pDonor has a TIR of pCMV-BG that drives the expression of mNeon fluorescent protein. pClpx is a pCMV-βGlobin promoter that drives the *E. coli* ClpX-NLS sequence. All three plasmids were transfected into HEK293T cells at a ratio of 12 µg pHelper: 6 µg pDonor: 1 µg pClpX: 54 µL Mirus-LT1 (transfection reagent). The cells were then incubated at 37°C for 72 hours, and their gDNA was collected.

[0119] Figure 56 Transposition of pDonor to the single-copy target AAVS1 was depicted. AAVS1 is a safe harbor locus that allows foreign genes to be expressed harmlessly in human cells when the cassette is integrated into the human genome. AAVS1 is presented only once on chromosome 19 in the human genome. By measuring the junctional PCR of genomic DNA collected from cells transfected with both AAVS1 target 5 and AAVS1 target 6 using pHelper, the transposition of pDonor to the intended target could be visualized in the forward LE direction.

[0120] Figure 57 Sanger sequencing of transposable AAVS1 targets 5 and 6 was depicted in the forward LE direction. The AAVS5 transposation had a first transposition 63 bp from PAM, and the AAVS1 target 6 had a first transposition event 61 bp from PAM.

[0121] Figure 58A and 58B The description of the target 5 in AAVS ( Figure 58A) and target 6 ( Figure 58B NGS sequencing of transposons performed at ) location.

[0122] Figure 59 A schematic diagram of NGS quantification is depicted. Hypothetical transposition is modeled on top of the unintegrated target sequence. The two primers should be as close as possible.

[0123] Figure 60 Transposition at AAVS1 target 5 is depicted. Transposition-specific primers were used to amplify six gDNA preparations of the AAVS1 target 5 pHelper (top inset), where transposition bands reflect the forward LE junction reaction from the genomic target to the donor cargo. Target-free pHelper refers to cultures for which the spacer sequence does not specify a target. Transposition bands are not visible under target-free pHelper conditions. Bottom inset: 25x cycle PCR amplification shows that the loading of the indexing step reaction for NGS sequencing was equal for both samples treated with the AAVS1 target 5 pHelper and those without target pHelper treatment.

[0124] Figures 61A-61C The NGS efficiency of unedited alleles and allele visualizations were depicted. Figure 61A This is a graph showing the NGS efficiency measurement results of AAVS1 target 5 compared to the control. Figure 61B Allelic mappings of unedited alleles in targetless NGS reads were depicted, showing small fractions that could be mapped to reference reads with SNPs. Figure 61C Allelic mappings of unedited alleles in the AAVS1 target 5 reads were depicted, showing similarity scores of reads indicating SNPs that have occurred at genomic loci, which are not due to AAVS1 target 5 incorporation into pHelper.

[0125] Figure 62 A schematic diagram of the plasmids used in the experiments of this disclosure is depicted.

[0126] Figures 63A-63B The integration of linearized donors into HEK293T cells was depicted. Figure 63A NGS integration of linear CAST pDonor without a single guide (pHelper empty) or with an AAVS1-5 target (pHelper AIO) is depicted. Figure 63B ddPCR integration of linear CAST pDonor without a single guide (pHelper empty) or with an AAVS1-5 target (pHelper AIO) was depicted.

[0127] Figure 64Integration of the plasmid donor into K562 cells was depicted. CAST pDonor, equipped with a one-way guide expression cassette, was used in cells containing 2 x 10⁻⁶ plasmid donors. 5 One (left) or 5 x 10 5 NGS integration in nuclear transfection of (right) K562 cells, wherein the single-guided expression cassette targets single-guided expression cassettes without helper plasmids (pHelper empty) or AAVS1-5 with AAVS1-5 target (pHelper AIO).

[0128] Figures 65A-65B Hep3B transposition using Lipofectamine 2000 and Lipofectamine 3000 is depicted, showing NGS quantification of pDonor containing sg cassette.

[0129] Figure 66 NGS detection of transposition in human albumin (ALB) via HEK293T was depicted. Guide targets 1077-1138 are predicted sites at albumin intron 1. 1139 and 1140 are guides constructed for AAVS1.

[0130] Figures 67A-67C Depicting a distance of 1093 from the target ( Figure 67A (Specifically, SEQ ID NO: 1264-1274, in order of appearance)), 1101 ( Figure 67B (Specifically, SEQ ID NO: 1275-1279, in order of appearance)), 1115 ( Figure 67C (Separately, SEQ ID NO: 1280-1281, in order of appearance) NGS read alignment of the modeled transposon target at PAM 60 bp.

[0131] Figure 68 NGS detection of transposons in mouse albumin (mALB) at Hepa1–6 was depicted. Guide targets 1141–1182 are predicted sites at intron 1 of mouse albumin.

[0132] Figures 69A-69C Depicting a distance of 1148 ( ) from the target Figure 69A (Specifically, SEQ ID NO: 1282-1285, in order of appearance)), 1161 ( Figure 69B (Specifically, SEQ ID NO: 1286-1288, in order of appearance) and 1162 ( Figure 69C NGS read alignment of the modeled transposon target at PAM 60 bp (separately, SEQ ID NO: 1289-1297, in order of appearance)

[0133] Figure 70 NGS detection of transposition in Hepa1-6 of mouse albumin (mROSA26) was depicted. Guide targets 1183-1167 are predicted sites at mRosa26.

[0134] Figures 71A-71E Depicting the distance from the target ( Figure 71A (Specifically, SEQ ID NO: 1298-1304, in order of appearance), 1201 ( Figure 71B (SEQ ID NO: 1305-1313, in order of appearance), 1205 ( Figure 71C (Specifically, SEQ ID NO:1314-1323, in order of appearance)), 1219 ( Figure 71D (Specifically, SEQ ID NO: 1324-1327, in order of appearance) and 1257 ( Figure 71E (Separately, SEQ ID NO: 1328-1330, in order of appearance) NGS read alignment of the modeled transposon target at PAM 60 bp.

[0135] Figure 72 NGS readouts for copy-increase single-guide conditions were depicted. Control conditions reflect all components expressed on the plasmid (Cas12k, S15, TniQ, TnsB, TnsC, and single-guide targeting AAVS1-5) in pHelper where the donor plasmid contains the fluorescent marker. For the remaining conditions, integration was quantified using FIX cargoes. The 0 x sg condition has no single-guide on the pHelper plasmid but has FIX pDonor. The 1 x sg condition has single-guide on the pHelper plasmid with pDonor FIX. The 2 x sg condition does not contain single-guide on the pHelper plasmid, where two single-guide cassettes target AAVS1-5 on the pDonor FIX plasmid. The 3 x sg condition contains pDonor FIX containing two single-guide cassettes targeting AAVS1-5, where the pHelper plasmid contains an additional single-guide cassette targeting AAVS1-5.

[0136] Figure 73 The ddPCR detection of both LE and RE binding sites for promoter driver factor IX (FIX) delivery was depicted under unguided (pHelper empty) and AAVS1 guided (pHelper AAVS5) conditions.

[0137] Figure 74The tree depicts MG161 family members as distant homologs of sso7d. The tree was inferred from multiple sequence alignments of full-length protein sequences containing PFamPF02294 domain hits. Reference sso7d sequences are highlighted with triangles. The distance between apexes is estimated at 0.5 substitutions per site (bars).

[0138] Figures 75A-75C depict the functional domain of MG161 encoding a tandem repeat sequence. Figure 75A shows the genomic background of proteins encoding multiple functional domains (FDs). FDs correspond to tandem incomplete repeat sequences (arrows labeled 161-12 to 161-18). Figure 75B shows a multiple sequence alignment of the tandem repeat sequence FDs with the reference sso7d sequence from *S. solfatarcica* (SEQ ID NO: 1331-1339, respectively, in order of appearance). MG161-13 has 20% AAI with the reference sequence, while the other FDs have lower sequence identity. Figure 75C The results show that the 3D structure prediction of the ORF encoding the repeating structural domains indicates that each structural domain forms a 4-β sheet-like structure connected by flexible joints.

[0139] Figure 76 The tree depicts MG162 family members as long-range homologs of HMGN1. The tree was inferred from multiple sequence alignments of full-length protein sequences containing the PFamPF01101 domain hit (SEQ ID NO: 1340-1353, respectively, in order of appearance). Reference HMGN1 sequences are highlighted with triangles. The distance between the apexes is estimated at 0.7 substitutions (bars) per site.

[0140] Figure 77 Multiple sequence alignments of the MG162 functional domain protein with reference human and mouse HMGN1 sequences were depicted. The mean pairwise identity percentage was 40.4%. The conserved RXSXRLS motif (SEQ ID NO: 1255) is highlighted with a black box.

[0141] Figures 78A-78B The TniQ test of the fused functional structural domains is described. Figure 78A A library containing the functional domains was shown, which was introduced into a TniQ expression vector and expressed separately with a pHelper vector containing other components required for transposition (Cas12k, S15, TnsB, TnsC, and a single guide targeting AAVS1-5). The wild-type H1 nuclear TniQ fusion and the pHelper split vector control were undetectable. Figure 78BThe top 14 most active functional domains from the split vector test, retested in single-expression constructs, are shown. Of all constructs tested, 13 of the 14 functional domains exhibited higher transposition efficiency in the single-expression setting compared to the WT single-expression control. Brief description of sequence lists

[0142] The sequence listing submitted herein provides exemplary polynucleotide and polypeptide sequences for use in methods, compositions, and systems according to this disclosure. An exemplary description of the sequences therein is given below.

[0143] MG64 SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85 and 220 show the full-length peptide sequence of the MG64 Cas effector.

[0144] SEQ ID NO: 2-4, 13-15, 17-19, 65-67 and 109-111 show peptide sequences of MG64 transposable proteins, which may include recombinase / transposase recognition complexes associated with MG64 Cas effectors.

[0145] SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122 and 222 show the nucleotide sequences of MG64 tracrRNA derived from the same locus as the MG64 Cas effector.

[0146] SEQ ID NO: 7 and 34-35 show the nucleotide sequences of the MG64 target CRISPR repeat sequence.

[0147] SEQ ID NO: 106-108, 112-118 and 221 show the nucleotide sequences of MG64 crRNA.

[0148] SEQ ID NO: 8, 10, 39-44, 77, 79 and 93 show the nucleotide sequences of the right-hand transposase recognition sequence associated with the MG64 system.

[0149] SEQ ID NO: 9, 11, 36-38, 76 and 78 show the nucleotide sequences of the left transposase recognition sequence associated with the MG64 system.

[0150] SEQ ID NO: 45-63, 68-75, 96-103 and 123-140 show the nucleotide sequences of single guide RNAs engineered to function in conjunction with the MG64 Cas effector.

[0151] SEQ ID NO: 208 shows the nucleotide sequence of the MG64 expression construct.

[0152] SEQ ID NO: 223 shows the nucleotide sequence of the MG64 active donor.

[0153] SEQ ID NO: 228-230 shows the full-length peptide sequence of the MG64 accessory protein.

[0154] SEQ ID NO: 233-234 shows the nucleotide sequence of the MG64 target site.

[0155] SEQ ID NO: 369-371 shows the nucleotide sequence of the MG64 active target sequence.

[0156] MG190 SEQ ID NO: 209-219 shows the full-length peptide sequence of the MG190 ribosomal protein S15 homolog.

[0157] Other sequences SEQ ID NO: 86-87, 192-207 and 1354-1383 show the peptide sequences of the nuclear localization signal.

[0158] SEQ ID NO: 88-89 shows the peptide sequence of the linker.

[0159] SEQ ID NO: 90-92 shows the peptide sequences of the epitope tag.

[0160] SEQ ID NO: 141-143 shows the genomic target sequences.

[0161] SEQ ID NO: 144-180 shows the target guidance sequence.

[0162] SEQ ID NO: 181-183 shows the nucleic acid sequence of the S15 fusion protein.

[0163] SEQ ID NO: 184 shows the donor construct.

[0164] SEQ ID NO: 185 shows the MG64-1 sgRNA sequence.

[0165] SEQ ID NO: 186 shows the connector sequence.

[0166] SEQ ID NO: 187-189 shows the amino acid sequence of the S15 fusion protein.

[0167] SEQ ID NO: 190-191 shows the promoter sequences.

[0168] SEQ ID NO: 224-226 and 231-232 show the nucleotide sequences of the primers.

[0169] SEQ ID NO: 227 shows the nucleotide sequence of the plasmid element.

[0170] SEQ ID NO: 235-249 shows the peptide sequence of the ClpX accessory protein.

[0171] SEQ ID NO: 250-251 shows the nucleotide sequence of the primer.

[0172] SEQ ID NO: 252 shows the nucleotide sequence of the plasmid element.

[0173] SEQ ID NO: 253 shows the nucleotide sequence of the primer.

[0174] SEQ ID NO: 254-256 shows the nucleotide sequences of the MG64 active target sequence.

[0175] SEQ ID NO: 257-282 and 1138-1241 show the protein sequences of the MG161 functional domain.

[0176] SEQ ID NO: 283-307 and 1242-1254 show the protein sequences of the MG162 functional domain.

[0177] SEQ ID NO: 308-359 shows the protein sequences of the H1 nuclear library.

[0178] SEQ ID NO: 360-368 and 372-753 show the nucleotide sequences of the primers and probes.

[0179] SEQ ID NO: 754-944 shows the nucleotide sequence of the unidirectional target.

[0180] SEQ ID NO: 945-1135 shows the nucleotide sequence of the primer binding sequence.

[0181] SEQ ID NO: 1136 shows the nucleotide sequence of the promoter.

[0182] SEQ ID NO: 1137 shows the protein sequence of the expression construct. Detailed Implementation

[0183] While various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Various changes, modifications, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0184] Unless otherwise indicated, the practice of some of the methods disclosed herein employs techniques from immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See, for example, Sambrook and Green et al., *Molecular Cloning: A Laboratory Manual*, 4th edition (2012); *Current Protocols in Molecular Biology* (edited by FM Ausubel et al.); *Methods in Enzymology* (Academic Press, Inc.), *PCR 2: A Practical Approach* (edited by MJ MacPherson, BD Hames, and GR Taylor (1995)); *Antibodies, A Laboratory Manual* (edited by Harlow and Lane (1988)); and *Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications*, 6th edition (edited by RI Freshney (2010)).

[0185] As used herein, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” are intended to include the plural forms as well. Furthermore, where the terms “including,” “include,” “having,” “has,” “with,” or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0186] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value, as determined by a person of ordinary skill in the art, which will depend in part on how the value was measured or determined, i.e., the limitations of the measurement system. For example, “about” may mean within one or more standard deviations according to practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0187] As used herein, the term "nucleotide" refers to a base-sugar-phosphate combination. Contemplated nucleotides include naturally occurring nucleotides and synthetic nucleotides. A nucleotide is a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide includes ribonucleoside triphosphates, adenosine triphosphates (ATP), uridine triphosphates (UTP), cytosine triphosphates (CTP), guanosine triphosphates (GTP), and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [αS]dATP, 7-denitro-dGTP, and 7-denitro-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide encompasses dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of ddNTPs include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled, such as using portions containing an optically detectable portion (e.g., a fluorophore) or quantum dots. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), Waterfall Blue, Oregon Green, Texas Red, Cyanide, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, available from Perkin Elmer, Foster City, California; and FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, and FluoroLink Fluor, available from Amersham, Arlington Heights, IL. X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP; fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP, available from Boehringer Mannheim, Indianapolis, Ind.; and Molecular Probes, Eugene, Oregon, available from Molecular Probes, Eugene, Oregon. Chromosome-tagged nucleotides obtained from Oregon, including BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Waterfall Blue-7-UTP, Waterfall Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP. The term nucleotide encompasses chemically modified nucleotides. An exemplary chemically modified nucleotide is biotin-dNTP.Non-limiting examples of biotinylated dNTPs include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0188] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to polymeric forms of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or their analogues, in single-stranded, double-stranded, or multi-stranded form. Contemplated polynucleotides include genes or segments thereof. Exemplary polynucleotides include, but are not limited to, DNA, RNA, coding or non-coding regions of genes or gene segments, multiple loci (locus) as defined by ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-free polynucleotides containing cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. In polynucleotides, when T is mentioned, T refers to U (uracil) in RNA and T (thymine) in DNA. Polynucleotides can be exogenous or endogenous to cells and / or present in cell-free environments. The term polynucleotide encompasses modified polynucleotides (e.g., altered backbone, sugar, or nucleobase). Modifications to the nucleotide structure are conferred, if present, before or after polymer assembly. Non-limiting examples of modifications include: 5-bromouracil, peptide nucleic acids, heteronucleotides, morpholinonucleotides, locked nucleic acids, glycerol nucleic acids, threonine nucleic acids, dideoxynucleotides, cordycepin, 7-denitro-GTP, fluorophores (e.g., sugar-linked rhodamine or fluorescein), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, piracetamidine, and woyoside. The sequence of a nucleotide can be interrupted by non-nucleotide components.

[0189] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to polymers of at least two amino acid residues linked by peptide bonds. This terminology does not indicate a specific length of the polymer, nor is it intended to imply or distinguish whether a peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally occurring. The terminology applies to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer may contain non-amino acid components. The terminology includes amino acid chains of any length, including full-length proteins and proteins with or without secondary or tertiary structures (e.g., domains). The terminology also covers modified amino acid polymers; for example, through disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, oxidation, and any other manipulation, such as conjugation with labeled components. As used herein, the term “amino acid” refers to both natural and non-natural amino acids, including, but not limited to, modified amino acids. Modified amino acids include natural amino acids that have been chemically modified to include groups or chemical moieties on the amino acid that are not naturally occurring. The term “amino acid” includes both D-amino acids and L-amino acids.

[0190] As used herein, “non-natural” refers to a nucleic acid or polypeptide sequence that is not naturally occurring. Non-natural means a nucleic acid or polypeptide sequence that contains modifications such as mutations, insertions, or deletions. The term non-natural encompasses fused nucleic acids or polypeptides that encode or exhibit the activity (e.g., enzyme activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) of a nucleic acid or polypeptide sequence fused with a non-natural sequence. Non-natural nucleic acid or polypeptide sequences include those that have been genetically engineered to link with naturally occurring nucleic acid or polypeptide sequences (or variants thereof) to produce chimeric nucleic acids or polypeptide sequences encoding chimeric nucleic acids or polypeptides.

[0191] As used herein, “operationally linked,” “operationally connected,” “operationally connected,” or their syntactic equivalents refer to the arrangement of genetic elements, such as promoters, enhancers, polyadenylated sequences, etc., in which an operation (e.g., movement or activation) of a first genetic element has some effect on a second genetic element. The effect on the second genetic element can be, but does not have to be, the same type of operation as that of the first genetic element. For example, if movement of the first element results in activation of the second element, the two genetic elements are operably linked. For example, if a regulatory element contributes to initiating transcription of a coding sequence, a regulatory element containing promoter and / or enhancer sequences can be operably linked to a coding region. Insertion residues may exist between the regulatory element and the coding region as long as this functional relationship is maintained.

[0192] A “functional segment” of a DNA or protein sequence is a segment that retains biological activity (function or structure) substantially similar to that of the full-length DNA or protein sequence. The biological activity of a DNA sequence includes its ability to influence expression in a manner attributable to the full-length sequence.

[0193] The terms “engineered,” “synthetic,” and “artificial” are used interchangeably herein to refer to objects modified through human intervention. For example, these terms refer to non-naturally occurring polynucleotides or polypeptides. Engineered peptides have, but do not need to have, low sequence identity with naturally occurring human proteins (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity). For example, the VPR and VP64 domains are synthetic trans-activation domains. Non-limiting examples include: nucleic acids being modified by altering their sequence to a sequence not found in nature; nucleic acids being modified by ligating them to nucleic acids that do not associate with them in nature, such that the ligation product has a function not present in the original nucleic acid; engineered nucleic acids being synthesized in vitro with sequences not found in nature; proteins being modified by altering the amino acid sequence of a protein to a sequence not found in nature; and engineered proteins acquiring new functions or properties. An “engineered” system includes at least one engineered component.

[0194] The terms "tracrRNA" or "tracr sequence" refer to trans-activating CRISPR RNA. tracrRNA interacts with CRISPR(cr)RNA to form guide (g)RNA in type II and subtype VB CRISPR-Cas systems. If the tracrRNA is engineered, it can be coupled with a wild-type exemplary tracrRNA sequence (e.g., from Streptococcus pyogenes). S. pyogenes Staphylococcus aureus ( S. aureusThe tracrRNA has approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% sequence identity and / or sequence similarity. tracrRNA can refer to a modified form of tracrRNA, which may include nucleotide changes such as deletions, insertions or substitutions, variants, mutations, or chimeras. The term tracrRNA encompasses a nucleic acid sequence that is at least approximately 60% identical to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.) for at least six consecutive nucleotides. For example, the tracrRNA sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical for at least six consecutive nucleotides of a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.). Type II tracrRNA sequences can be predicted on the genome sequence by identifying regions complementary to a portion of a repetitive sequence in an adjacent CRISPR array.

[0195] As used herein, “guide nucleic acid” or “guide polynucleotide” refers to a nucleic acid that can hybridize with a target nucleic acid and thereby guide an associated nuclease to the target nucleic acid. A guide nucleic acid is, but is not limited to, RNA (guide RNA or gRNA), DNA, or a mixture of RNA and DNA. A guide nucleic acid may include crRNA or tracrRNA or a combination of both. The term guide nucleic acid encompasses engineered guide nucleic acids and programmable guide nucleic acids for specific binding to a target nucleic acid. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. The strand of a double-stranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid is the complementary strand. The strand of a double-stranded target polynucleotide that is complementary to the complementary strand and therefore not complementary to the guide nucleic acid is called the non-complementary strand. A guide nucleic acid having a polynucleotide chain is a “single guide nucleic acid.” A guide nucleic acid having two polynucleotide chains is a “double guide nucleic acid.” Unless otherwise stated, the term “guide nucleic acid” is inclusive and refers to both single and double guide nucleic acids. A guide nucleic acid may contain a segment referred to as a “nucleic acid targeting segment,” “nucleic acid targeting sequence,” or “spacer.” Nucleic acid targeting regions may contain sub-regions, which are referred to as “protein-binding regions” or “protein-binding sequences” or “Cas protein-binding regions”.

[0196] In the context of two or more nucleic acid or polypeptide sequences, the terms “sequence identity” or “percentage identity” refer to two (e.g., in paired alignments) or more (e.g., in multiple sequence alignments) sequences that are identical or have the same specific percentage of amino acid residues or nucleotides when compared and aligned within a local or global comparison window to obtain maximum correspondence, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for peptide sequences include, for example: BLASTP for peptide sequences longer than 30 residues, using a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix with a gap penalty of 11 for presence, 1 for expansion, and conditional composition of the scoring matrix; BLASTP for sequences shorter than 30 residues, using a word length (W) of 2, an expected value (E) of 1000000, and a PAM30 scoring matrix with a gap penalty of 9 for gap opening and 1 for expansion (these are the default parameters for BLASTP in the BLAST suite, available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW using the Smith-Waltman homology search algorithm with a match of 2, a mismatch of -1, and a gap of -1; MUSCLE using default parameters; MAFFT using a retree of 2 and a maximum iteration of 1000; Novafold using default parameters; and HMMER hmmalign using default parameters.

[0197] This disclosure includes variants of any of the enzymes described herein that have one or more conserved amino acid substitutions. Such conserved substitutions can occur in the amino acid sequence of a polypeptide without disrupting the polypeptide's three-dimensional structure or function. Conservative substitutions can be accomplished by substituting amino acids with similar hydrophobicity, polarity, and R-chain lengths into each other. Alternatively or additionally, conserved substitutions can be identified by comparing aligned sequences of homologous proteins from different species, by locating mutated amino acid residues between species (e.g., non-conserved residues) without altering the fundamental function of the encoded protein. Such conserved substitution variants may include variants having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of the systems described herein (e.g., the MG64 system described herein). In some embodiments, such conserved substitution variants are functional variants. Such functional variants may cover sequences with substitutions such that the activity of key active site residues of the endonuclease is not disrupted. In some embodiments, functional variants of any of the systems described herein lack Figure 4A , 4B Substitution of at least one of the conserved or functional residues shown in 5. In some embodiments, functional variants of any system described herein lack Figure 4A , 4B The substitution of all of the conserved residues or functional residues shown in 5.

[0198] This disclosure also includes variants of any enzyme described herein that replace one or more catalytic residues to reduce or eliminate enzyme activity (e.g., reduced-activity variants). In some embodiments, a reduced-activity variant of the protein described herein comprises a disruptive substitution of at least one, at least two, or all three catalytic residues.

[0199] Conserved substitutions of functionally similar amino acids can be obtained from various references (see, for example, Creighton, *Proteins: Structures and Molecular Properties* (WH Freeman & Co.; 2nd ed. (December 1993))). The following eight groups each contain amino acids that are conserved in their substitutions: 1) Alanine (A), glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M).

[0200] As used herein, the term "RuvC_III domain" refers to the third discontinuous segment of the RuvC endonuclease domain (the RuvC nuclease domain comprises three discontinuous segments: RuvC_I, RuvC_II, and RuvC_III). RuvC domains or segments thereof can typically be identified by alignment with recorded domain sequences, by alignment with the structures of proteins with annotated domains, or by comparison with a Hidden Markov Model (HMM) constructed based on recorded domain sequences (e.g., Pfam HMM PF18541 for RuvC_III).

[0201] As used herein, the term "HNH domain" refers to a nuclease domain containing characteristic histidine and asparagine residues. HNH domains can typically be identified by comparison with recorded domain sequences, with the structure of proteins containing annotated domains, or by comparison with a hidden Markov model (HMM) constructed based on recorded domain sequences (e.g., Pfam HMM PF01844 for the HNH domain).

[0202] As used herein, the term “recombinase” refers to an enzyme that mediates the recombination of DNA fragments located between recombinase recognition sequences, resulting in the excision, insertion, inversion, exchange, or translocation of DNA fragments located between recombinase recognition sequences.

[0203] As used herein, in the context of nucleic acid modification (e.g., genome modification), the term "recombine" or "recombination" refers to the process by which two or more nucleic acid molecules, or two or more regions of a single nucleic acid molecule, are modified by the action of recombinase proteins. Recombination can particularly result in the excision, insertion, inversion, exchange, or translocation of nucleic acid sequences, for example, within or between one or more nucleic acid molecules.

[0204] As used herein, the term "transposon" or "transposable element" refers to a nucleic acid sequence in the genome that is a mobile genetic element capable of altering its location within the genome. In some embodiments, transposons transport additional "cargo DNA" excised from the genome. Transposons include, for example, retrotransposons, DNA transposons, autonomous and non-autonomous transposons, and class III transposons. The nucleic acid sequence of a transposon includes, for example, a gene encoding a homologous transposase, one or more transposase recognition sequences, or a combination thereof. In some embodiments, these transposons differ in the type of nucleic acid to be transposed, the type of repetitive sequence located at the end of the transposon, the type of cargo to be carried, or the transposition mode (i.e., self-repair or host repair). As used herein, the term "transposase" refers to an enzyme that binds to the recognition sequence of a transposon and catalyzes its movement to another part of the genome. In some embodiments, movement is carried out via a cut-and-paste mechanism or a replication transposition mechanism.

[0205] As used herein, the term "Tn7" or "Tn7-like transposase" refers to the transposase family, which comprises three main components: heteropolymer transposases (TnsA and / or TnsB) and regulatory proteins (TnsC). In addition to the TnsABC transposases, Tn7 elements can encode specialized target-site selection proteins TnsD and TnsE. Upon binding to TnsABC, the sequence-specific DNA-binding protein TnsD guides transposition to what is known as the "Tn7 attachment site," a conserved site of attTn7. TnsD is a member of a large family of proteins that also includes TniQ. TniQ has been shown to target transpossession at plasmid resolution sites.

[0206] As used herein, the term "complex" refers to the connection of at least two components. Each of the two components may retain its properties / activities prior to the formation of the complex. The connection can be achieved through covalent bonding, non-covalent bonding (i.e., hydrogen bonding, ionic interactions, van der Waals interactions, and hydrophobic bonds), the use of linkers, fusion, or any other suitable method. In some embodiments, the components of the complex are polynucleotides, polypeptides, or combinations thereof. For example, a complex may include a Cas protein and a guide nucleic acid.

[0207] In some embodiments, the CAST system described herein includes one or more Tn7 or Tn7-like transposases. In some example embodiments, the Tn7 or Tn7-like transposases comprise multimeric protein complexes. In some example embodiments, the multimeric protein complexes comprise TnsA, TnsB, TnsC, or TniQ. In these combinations, the transposases (TnsA, TnsB, TnsC, TniQ) may form complexes or fusion proteins with each other.

[0208] In some embodiments, the CAST system described herein includes one or more Tn5053 or Tn5053-like transposases. In some example embodiments, the Tn5053 or Tn5053-like transposases comprise multimeric protein complexes. In some example embodiments, the multimeric protein complexes comprise TnsA, TnsB, TnsC, or TniQ. In these combinations, the transposases (TnsA, TnsB, TnsC, TniQ) may form complexes or fusion proteins with each other.

[0209] As used herein, the term "Cas12k" (or alternatively, "type 2 VK") refers to a subtype of a type V CRISPR system that has been found to be defective in nuclease activity (e.g., it may include at least one defective RuvC domain lacking at least one catalytic residue important for DNA cleavage). This effector subtype is typically associated with the CAST system.

[0210] According to IUPAC conventions, the following abbreviations are used throughout the instance: A = adenine C = cytosine G = Guanine T = thymine R = adenine or guanine Y = cytosine or thymine S = guanine or cytosine W = adenine or thymine K = guanine or thymine M = adenine or cytosine B = C, G, or T D = A, G, or T H = A, C, or T V = A, C, or G Overview The discovery of novel Cas enzymes with unique functions and structures may offer the potential to further disrupt DNA editing technologies, thereby improving speed, specificity, functionality, and ease of use. Compared to the predicted prevalence of clustered regularly spaced short palindromic repeat (CRISPR) systems in microorganisms and the sheer diversity of microbial species, relatively few functionally characterized CRISPR / Cas enzymes exist in the literature. This is partly because a large number of microbial species may not be easily cultured under laboratory conditions. Metagenomic sequencing of natural environmental niches representing a large number of microbial species could provide a significant increase in the number of newly recorded CRISPR / Cas systems and accelerate the discovery of novel oligonucleotide editing functions. A recent example of the fruitfulness of this approach is demonstrated by the discovery of the CasX / CasY CRISPR system in 2016 through metagenomic analysis of a natural microbial community.

[0211] The CRISPR / Cas system is an RNA-guided nuclease complex that has been described as acting as an adaptive immune system in microorganisms. In its natural environment, the CRISPR / Cas system occurs within CRISPR (clustered, regularly spaced short palindromic repeats) operons or loci, typically comprising two parts: (i) an array of short repeat sequences (30-40 bp) separated by equally short spacer sequences, encoding RNA-based targeting elements; and (ii) an ORF encoding Cas, which encodes a nuclease polypeptide guided by the RNA-based targeting element along with an accessory protein / enzyme. Efficient nuclease targeting of a specific target nucleic acid sequence usually requires both: (i) complementary hybridization between the first 6-8 nucleic acids of the target (target seed) and the crRNA guide; and (ii) the presence of a protospacer adjacent motif (PAM) sequence (PAMs are typically sequences not commonly represented within the host genome) near the definition of the target seed. Based on their exact function and organization, CRISPR-Cas systems are typically classified into 2 categories, 5 types, and 16 subtypes based on shared functional characteristics and evolutionary similarity (see [link to relevant documentation]). Figure 1 ).

[0212] Class 1 CRISPR-Cas systems have large multi-subunit effector complexes and include types I, III, and IV.

[0213] Type I CRISPR-Cas systems are considered moderately complex in terms of their components. In a type I CRISPR-Cas system, an array of RNA-targeting elements is transcribed into long precursor crRNA (precursor crRNA / pre-crRNA), which is processed at repeating elements to release short mature crRNA. This short mature crRNA, followed by a suitable short concordant sequence called the protospacer neighbor motif (PAM), guides the nuclease complex to the nucleic acid target. This processing is carried out via an endonuclease subunit (Cas6) of a large endonuclease complex called a cascade, which also includes a crRNA-guided nuclease complex nuclease protein component (Cas3). Cas I nucleases primarily function as DNA nucleases.

[0214] The type III CRISPR system is characterized by the presence of a central nuclease called Cas10 and a repeat-associated mystery protein (RAMP) including Csm or Cmr protein subunits. As in the type I system, mature crRNA is processed from the crRNA precursor using a Cas6-like enzyme. Unlike the type I and type II systems, the type III system appears to target and cleave the DNA-RNA duplex (such as the DNA strand used as a template for RNA polymerase).

[0215] Type IV CRISPR-Cas systems have effector complexes comprising two genes of RAMP proteins from the highly reduced large subunit nuclease (csf1), Cas5 (csf3), and Cas7 (csf2) groups, as well as genes of the predicted small subunit in some embodiments; such systems are typically found on endogenous plasmids.

[0216] Class 2 CRISPR-Cas systems typically have single-peptide multi-domain nuclease effectors and include types II, V, and VI.

[0217] Type II CRISPR-Cas systems are considered the simplest in terms of components. In Type II CRISPR-Cas systems, processing the CRISPR array into mature crRNA does not require the presence of a specific endonuclease subunit; instead, it requires a small trans-coding crRNA (tracrRNA) whose region is complementary to the array repetitive sequence. The tracrRNA interacts with its corresponding effector nuclease (e.g., Cas9) and the repetitive sequence to form a precursor dsRNA structure. This precursor dsRNA structure is cleaved by endogenous RNase III, generating a mature effector enzyme loaded with both tracrRNA and crRNA. Type II nucleases are called DNA nucleases. Type II effectors typically exhibit a structure composed of RuvC-like endonuclease domains that employ RNase H folds, in which an unrelated HNH nuclease domain is inserted. The RuvC-like domain is responsible for cleaving the target (e.g., crRNA complementary) DNA strand, while the HNH domain is responsible for cleaving the replaced DNA strand.

[0218] Type V CRISPR-Cas systems are characterized by nuclease effector structures (e.g., Cas12) that are structurally similar to type II effectors, which include RuvC-like domains. Similar to type II, most (but not all) type V CRISPR systems use tracrRNA to process precursor crRNA into mature crRNA; however, unlike type II systems which require RNase III to cleave the precursor crRNA into multiple crRNAs, type V systems are able to use the effector nuclease itself to cleave the precursor crRNA. Like type II CRISPR-Cas systems, type V CRISPR-Cas systems are again referred to as DNA nucleases. Unlike type II CRISPR-Cas systems, some type V enzymes (e.g., Cas12a) appear to possess robust single-stranded nonspecific deoxyribonuclease activity activated by the directed cleavage of the first crRNA from the double-stranded target sequence.

[0219] Type VI CRISPR-Cas systems possess RNA-guided RNA endonucleases. The single polypeptide effector of a type VI system (e.g., Cas13) comprises two HEPN ribonuclease domains, rather than a RuvC-like domain. Unlike type II and V systems, type VI systems do not appear to require tracrRNA to process the precursor crRNA into crRNA. However, similar to type V systems, some type VI systems (e.g., C2C2) appear to possess robust single-stranded nonspecific nuclease (ribonuclease) activity activated by the first crRNA-directed cleavage of the target RNA.

[0220] Due to its simpler architecture, the Class 2 CRISPR-Cas has been most widely used in engineering and development as a designer nuclease / genome editing application.

[0221] This system is used for one of the early adaptations in vitro using (i) recombinant expression and purification of full-length Cas9 (e.g., class 2, type II Cas enzyme) isolated from Streptococcus pyogenes SF370; (ii) purified mature approximately 42 nt crRNA carrying an approximately 20 nt 5' sequence complementary to the target DNA sequence to be cleaved, followed by a 3' tracr binding sequence (the entire crRNA was transcribed in vitro from a synthetic DNA template carrying a T7 promoter sequence); (iii) purified tracrRNA transcribed in vitro from a synthetic DNA template carrying a T7 promoter sequence; and (iv) Mg 2+ A later-improved engineered system involves (ii) crRNA being linked to the 5' end of (iii) via a linker (e.g., GAAA) to form a single fusion synthetic guide RNA (sgRNA) capable of guiding Cas9 to its target (compare). Figure 2 (Top and bottom thumbnails).

[0222] Such engineered systems can be adapted for mammalian cells by providing DNA vectors encoding the following: (i) an ORF encoding a codon-optimized Cas9 (e.g., type II Cas enzyme) with a suitable mammalian promoter having a C-terminal nuclear localization sequence (e.g., SV40 NLS) and a suitable polyadenylation signal (e.g., TK pA signal); and (ii) an ORF encoding an sgRNA (with a 5' sequence starting with G followed by a 20 nt complementary targeting nucleic acid sequence linked to a 3' tracr binding sequence, an adapter, and a tracrRNA sequence) with a suitable polymerase III promoter (e.g., U6 promoter).

[0223] Transposons are mobile elements that can move between locations within the genome. These transposons have evolved to limit their negative effects on the host. Various regulatory mechanisms are used to maintain low-frequency transposition and sometimes coordinate transposition with various cellular processes. Some prokaryotic transposons can also mobilize functions beneficial to the host or otherwise help maintain the element's function. Certain transposons may also have evolved strict control mechanisms over target site selection; the most prominent example is the Tn7 family.

[0224] Transposon Tn7 and similar elements may be a reservoir of functions related to antibiotic resistance and pathogenesis in the clinical setting, as well as encoding other adaptive functions in the natural environment. For example, the Tn7 system has evolved mechanisms to almost completely avoid integration into important host genes, but also maximizes the dispersal of the element by recognizing mobile plasmids and bacteriophages that can move Tn7 between host bacteria.

[0225] Tn7 and Tn7-like elements can control the location and timing of their insertion, possessing one pathway that guides insertion into a single conserved location within the bacterial genome and a second pathway that appears well-suited for maximally targeting mobile plasmids capable of transporting elements between bacteria (see [link to relevant documentation]). Figure 3 The association between Tn7-like transposons and the CRISPR-Cas system suggests that transposons may hijack CRISPR effectors to generate R loops at target sites and facilitate the spread of transposons via plasmids and phages.

[0226] MG64 system In some embodiments, this document provides the MG64 system for transposing cargo nucleotide sequences into target nucleic acid sites. See also Figures 4A-4B .

[0227] In some embodiments, this document describes a system for transposing a cargo nucleotide sequence into a target nucleic acid site, the system comprising: a) a Cas effector complex comprising two types V Cas effectors, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide configured to hybridize with the target nucleic acid site; b) a Tn7 transposase complex configured to bind to the Cas effector complex and comprising a TnsB component, a TnsC component, and a TniQ component, and an accessory protein; and c) a double-stranded nucleic acid configured to interact with the Tn7 transposase complex and comprising the cargo nucleotide sequence.

[0228] In some aspects, this disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a Cas effector complex comprising two types V Cas effectors, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide that hybridizes to the target nucleic acid site; a Tn7 transposase complex that binds to the Cas effector complex and comprises a TnsB component, a TnsC component, and a TniQ component, and an accessory protein comprising a sequence having at least 70% sequence identity with any one of SEQ ID NO: 228-230 and 235-249; and a double-stranded nucleic acid that interacts with the Tn7 transposase complex and comprises the cargo nucleotide sequence.

[0229] In another aspect, this disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site, the system comprising: a Cas effector complex including two types V Cas effectors, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide that hybridizes to the target nucleic acid site; a Tn7 transposase complex that binds to the Cas effector complex and includes a functional domain (FD)-TniQ fusion and an accessory protein; and a double-stranded nucleic acid that interacts with the Tn7 transposase complex and includes the cargo nucleotide sequence.

[0230] In another aspect, this disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a Cas effector complex comprising two types V Cas effectors, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide, the engineered guide polynucleotide hybridizing to the target nucleic acid site; a Tn7 transposase complex, the Tn7 transposase complex binding to the Cas effector complex and comprising a functional domain (FD)-TniQ fusion and an accessory protein, wherein the functional domain (FD) comprises a sequence having at least 80% identity with any one of SEQ ID NO: 257-307 and 1138-1242; and a double-stranded nucleic acid interacting with the Tn7 transposase complex and comprising the cargo nucleotide sequence.

[0231] In some aspects, this disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a Cas effector complex comprising two class V Cas effectors, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide that hybridizes to the target nucleic acid site; a Tn7 transposase complex that binds to the Cas effector complex and comprises a TnsB component, a TnsC component, and a TniQ component, and an accessory protein comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 228-230 and 235-249; and a double-stranded nucleic acid that interacts with the Tn7 transposase complex and comprises the cargo nucleotide sequence.

[0232] In some embodiments, the system includes a double-stranded nucleic acid comprising a cargo nucleotide sequence. In some embodiments, this cargo nucleotide sequence interacts with a Tn7 or Tn5053 transposase complex. In some embodiments, the system includes a Cas effector complex. In some embodiments, the Cas effector complex includes two types of V Cas effectors and an engineered guide polynucleotide configured to hybridize with a target nucleotide sequence. In some embodiments, the system includes a Tn7 or Tn5053 transposase complex configured to bind to the Cas effector complex, wherein the Tn7 or Tn5053 transposase complex includes a TnsB subunit.

[0233] In some embodiments, the cargo nucleotide sequence is flanked by a left-side transposase recognition sequence. In some embodiments, the cargo nucleotide sequence is flanked by a right-side transposase recognition sequence. In some embodiments, the cargo nucleotide sequence is flanked by both a left-side and a right-side transposase recognition sequence.

[0234] In some embodiments, the target nucleic acid includes a target nucleic acid site. In some embodiments, the target nucleic acid includes a PAM sequence compatible with the Cas effector complex adjacent to the target nucleic acid site. In some embodiments, the PAM sequence is located at the 3' position of the target nucleic acid site. In some embodiments, the PAM sequence is located at the 5' position of the target nucleic acid site.

[0235] In some embodiments, engineered guide polynucleotides are configured to bind to type 2 V Cas effectors. In some embodiments, type 2 V Cas effectors are type 2 VK effectors. In some embodiments, type 2 V Cas effectors comprise polypeptides having a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 70% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 75% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 80% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 85% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 90% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 91% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 92% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 93% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220.In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 94% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 95% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 96% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 97% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, type 2 V Cas effectors include polypeptides comprising sequences having at least about 98% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, type 2 V Cas effectors include polypeptides comprising sequences having at least about 99% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, type 2 V Cas effectors include polypeptides comprising sequences having 100% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220.

[0236] In some embodiments, the TnsB subunit comprises a polypeptide having a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 70% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 75% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 80% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 85% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 90% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 91% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 92% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 93% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 94% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 95% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 96% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 97% identity with SEQ ID NO: 2, 13, 17, and 65.In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 98% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 99% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having 100% identity with SEQ ID NO: 2, 13, 17, and 65.

[0237] In some embodiments, the functional domain (FD)-TniQ fusion comprises a polypeptide having a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide having the same sequence as SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having at least about 70% identity with SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having at least about 75% identity with SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having at least about 80% identity with SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having at least about 85% identity with SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having at least about 90% identity with SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having at least about 91% identity with SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having at least about 92% identity with SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having at least about 93% identity with SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having at least about 94% identity with SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having at least about 95% identity with SEQ ID NO: 257-307 and 1138-1242.In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having at least about 96% identity with SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having at least about 97% identity with SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having at least about 98% identity with SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having at least about 99% identity with SEQ ID NO: 257-307 and 1138-1242. In some embodiments, the FD-TniQ fusion comprises a polypeptide containing a sequence having 100% identity with SEQ ID NO: 257-307 and 1138-1242.

[0238] In some embodiments, the Tn7 transposase complex comprises at least one polypeptide, said at least one polypeptide comprising a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide comprising a sequence having at least about 70% identity with any of SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 75% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 80% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 85% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 90% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 91% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 92% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 93% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 94% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111.In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 95% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 96% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 97% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 98% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide having a sequence having at least about 99% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide having a sequence having 100% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111.

[0239] In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently comprising a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently comprising a sequence having at least about 70% sequence identity with any one of SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently comprising a sequence having at least 75% sequence identity with any one of SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 85% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 90% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 91% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111.In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 92% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 93% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 94% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 95% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 96% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 97% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence with at least about 98% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence with at least about 99% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence with 100% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111.

[0240] In some embodiments, the Tn7 transposase complex includes a helper protein. In some embodiments, the helper protein is ClpX.

[0241] In some embodiments, the accessory protein comprises a sequence having at least 80% sequence identity with any of SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 70% identity with any of SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 75% identity with SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 80% identity with SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 85% identity with SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 90% identity with SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 91% identity with SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 92% identity with SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 93% identity with SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 94% identity with SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 95% identity with SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 96% identity with SEQ ID NO: 228-230 and 235-249.In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 97% identity with SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 98% identity with SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having at least about 99% identity with SEQ ID NO: 228-230 and 235-249. In some embodiments, the accessory protein comprises a polypeptide comprising a sequence having 100% identity with SEQ ID NO: 228-230 and 235-249.

[0242] In some embodiments, the systems disclosed herein include at least one engineered guide polynucleotide, such as gRNA.

[0243] In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 70% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 75% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 80% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 85% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 90% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 91% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 92% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122 and 222.In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 93% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 94% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 95% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 96% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 97% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 98% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 99% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having 100% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222.

[0244] In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides that is identical to any one of SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides that has at least about 70% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides that has at least about 75% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 80% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185, consisting of at least about 46-80 consecutive nucleotides. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 85% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185, consisting of at least about 46-80 consecutive nucleotides. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 90% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185, consisting of at least about 46-80 consecutive nucleotides. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 91% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140 and 185, comprising at least about 46-80 consecutive nucleotides.In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 92% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185. In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 93% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185. In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 94% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185. In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 95% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185. In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 96% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185. In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 97% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 98% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 99% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having 100% identity with SEQ ID NO: 45-63, 68-75, 96-103, 123-140, and 185.

[0245] In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides that is identical to any one of SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 70% identity to at least about 46-80 consecutive nucleotides that is identical to SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 75% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944, consisting of at least about 46-80 consecutive nucleotides. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 80% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944, consisting of at least about 46-80 consecutive nucleotides. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 85% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140 and 754-944, consisting of at least about 46-80 consecutive nucleotides.In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 90% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944, consisting of at least about 46-80 consecutive nucleotides. In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 91% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944, consisting of at least about 46-80 consecutive nucleotides. In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 92% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 93% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 94% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 95% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 96% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 97% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944.In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 98% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 99% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides that is 100% identical to SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140 and 754-944.

[0246] In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence identical to any one of SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 70% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 75% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 80% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 85% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 90% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising sequences having at least about 91% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140 and 754-944.In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 92% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 93% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 94% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 95% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 96% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 97% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 98% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 99% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having 100% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944.

[0247] In some aspects, this disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site, the system comprising: a Cas effector complex that hybridizes to the target nucleic acid site and includes: i) two types of V Cas effectors, the two types of V Cas effectors including a sequence having at least 80% sequence identity with any one of SEQ ID NO: 1, 81, 82, 83 and 85; and ii) an engineered guide polynucleotide that has at least 80% sequence identity with any one of SEQ ID NO: 5, 6, 45-63, 68-75, 96-103, 123-140 and 754-944; a Tn7 transposase complex that binds to the Cas effector complex and includes a TnsB component, a TnsC component and a TniQ component, and an accessory protein, the TnsB component, the TnsC component or the TniQ component including a sequence having at least 80% sequence identity with any one of SEQ ID NO: 1, 81, 82, 83 and 85; and ii) an engineered guide polynucleotide that hybridizes to the target nucleic acid site and includes: i) two types of V Cas effectors, the two types of V Cas effectors including a sequence having at least 80% sequence identity with any one of SEQ ID NO: 1, 81, 82, 83 and 85; and ii) a Tn7 transposase complex that hybridizes to the target nucleic acid site and includes: i) ... The accessory protein comprises a sequence having at least 80% identity with any of 2-4, the accessory protein comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 228-230 and 235-249; and a double-stranded nucleic acid that interacts with the Tn7 type transposase complex and comprises, in 5' to 3' order: i) a left transposase recognition sequence comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 9, 11, 36, 37 and 38; ii) the cargo nucleotide sequence; and ii) a right transposase recognition sequence comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 8, 39-44 and 93.

[0248] In some aspects, this disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site, the system comprising: a Cas effector complex that hybridizes to the target nucleic acid site and comprising: i) two types of V Cas effectors, the two types of V Cas effectors comprising a sequence having at least 80% sequence identity with SEQ ID NO: 12; and iii) an engineered guide polynucleotide that has at least 80% identity with any one of SEQ ID NO: 32, 102, 104, and 107; a Tn7 transposase complex that binds to the Cas effector complex and comprises a TnsB component, a TnsC component, and a TniQ component, and an accessory protein, the TnsB component, the TnsC component, or the TniQ component comprising a sequence having at least 80% identity with any one of SEQ ID NO: 13-15, and the accessory protein comprising a sequence having at least 80% identity with any one of SEQ ID NO: 12; and iii) an engineered guide polynucleotide that has at least 80% sequence identity with any one of SEQ ID NO: 12; The sequence having at least 80% sequence identity among any of 228-230 and 235-249; and a double-stranded nucleic acid that interacts with the Tn7 type transposase complex and comprises, in 5' to 3' order: i) a left transposase recognition sequence comprising a sequence having at least 80% sequence identity with SEQ ID NO: 76; ii) the cargo nucleotide sequence; and iii) a right transposase recognition sequence comprising a sequence having at least 80% identity with SEQ ID NO: 77.

[0249] In some aspects, this disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site, the system comprising: a Cas effector complex that hybridizes to the target nucleic acid site and comprising: i) two types of V Cas effectors, the two types of V Cas effectors comprising a sequence having at least 80% sequence identity with SEQ ID NO: 16; and ii) an engineered guide polynucleotide that has at least 80% identity with any one of SEQ ID NO: 33, 103, 105, and 108; a Tn7 transposase complex that binds to the Cas effector complex and comprises a TnsB component, a TnsC component, and a TniQ component, and an accessory protein, the TnsB component, the TnsC component, or the TniQ component comprising a sequence having at least 80% identity with any one of SEQ ID NO: 17-19, and the accessory protein comprising a sequence having at least 80% identity with any one of SEQ ID NO: 16; and ii) an engineered guide polynucleotide that has at least 80% sequence identity with any one of SEQ ID NO: 16; The sequence having at least 80% sequence identity among any one of 228-230 and 235-249; and a double-stranded nucleic acid that interacts with the Tn7 type transposase complex and comprises, in 5' to 3' order: i) a left transposase recognition sequence comprising a sequence having at least 80% sequence identity with SEQ ID NO: 78; ii) the cargo nucleotide sequence; and iii) a right transposase recognition sequence comprising a sequence having at least 80% sequence identity with SEQ ID NO: 79.

[0250] In some embodiments, the system further includes a PAM sequence compatible with the Cas effector complex. In some embodiments, the PAM sequence includes SEQ ID NO: 31.

[0251] In some embodiments, the PAM sequence is located at approximately 50 to approximately 70 base pairs from the target nucleic acid site. In some embodiments, the PAM sequence is located at the 3' position of the target nucleic acid site. In some embodiments, the PAM sequence is located at the 5' position of the target nucleic acid site.

[0252] In some embodiments, the guide RNA includes various structural elements, including, but not limited to: a spacer sequence that binds to the original spacer sequence (target sequence), crRNA, and optionally tracrRNA. In some embodiments, the guide RNA includes crRNA containing the spacer sequence. In some embodiments, the guide RNA further includes tracrRNA or modified tracrRNA.

[0253] In some embodiments, the system provided herein includes one or more guide RNAs. In some embodiments, the guide RNA includes a sense sequence. In some embodiments, the guide RNA includes an antisense sequence. In some embodiments, the guide RNA includes a nucleotide sequence other than a region complementary to or substantially complementary to a region of the target sequence. For example, crRNA is or is considered part of, or is included in, the guide RNA, such as a crRNA:tracrRNA chimera.

[0254] In some embodiments, the guide RNA comprises a synthetic nucleotide or a modified nucleotide. In some embodiments, the guide RNA comprises one or more nucleoside junctions modified from a natural phosphodiester. In some embodiments, all nucleoside junctions of the guide RNA or its sequential nucleotide sequence are modified. For example, in some embodiments, the nucleoside junctions comprise sulfur (S), such as phosphate thioester nucleoside junctions.

[0255] In some embodiments, the guide RNA includes modifications to the ribose sugar or nucleobase. In some embodiments, the guide RNA includes one or more nucleosides comprising a modified sugar moiety, wherein the modified glucose moiety is a modification of the sugar moiety compared to the ribose sugar moiety present in deoxyribonucleic acid (DNA) and RNA. In some embodiments, the modification is within a ribocycle structure. Exemplary modifications include, but are not limited to, substitution with a hexose ring (HNA), a bicyclic ring (e.g., locked nucleic acid (LNA)) having a biradical bridge between the C2 and C4 carbons on the ribocycle, or an unconnected ribocycle (e.g., UNA) typically lacking a bond between the C2 and C3 carbons. In some embodiments, the sugar-modified nucleoside comprises a bicyclic hexose nucleic acid or a tricyclic nucleic acid. In some embodiments, the modified nucleoside comprises a nucleoside in which the sugar moiety is replaced by a non-sugar moiety, such as a peptide nucleic acid (PNA) or a morpholino nucleic acid.

[0256] In some embodiments, the guide RNA comprises one or more modified sugars. In some embodiments, sugar modification comprises modification by changing a substituent on the ribose ring to a group other than hydrogen or a 2'-OH group naturally present in DNA and RNA nucleosides. In some embodiments, the substituent is introduced at the 2', 3', 4', or 5' position or a combination thereof. In some embodiments, the nucleoside having the modified sugar moiety comprises a 2'-modified nucleoside, such as a 2'-substituted nucleoside. In some embodiments, the 2'-sugar-modified nucleoside is a nucleoside having a substituent other than H or -OH at the 2' position (a 2'-substituted nucleoside) or comprising a 2'-linked diradical, and includes 2'-substituted nucleosides and LNA (2'-4' diradical-bridged) nucleosides. Examples of 2'-substituted modified nucleosides include, but are not limited to, 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. In some embodiments, the modification of the ribosome includes modification at the 2' position of the ribosome. In some embodiments, the modification at the 2' position of the ribosome is selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, and 2'-O-(2-methoxyethyl).

[0257] In some embodiments, the guide RNA comprises one or more modified sugars. In some embodiments, the guide RNA comprises only modified sugars. In some embodiments, the guide RNA comprises more than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises 2'-O-methoxyethyl. In some embodiments, the guide RNA comprises both nucleoside indirect head modification and nucleoside modification.

[0258] In some embodiments, the guide RNA includes a sequence complementary to a polynucleotide sequence of a eukaryotic, fungal, plant, mammalian, or human genome. In some embodiments, the guide RNA includes a sequence complementary to a eukaryotic genome polynucleotide sequence. In some embodiments, the guide RNA includes a sequence complementary to a fungal genome polynucleotide sequence. In some embodiments, the guide RNA includes a sequence complementary to a plant genome polynucleotide sequence. In some embodiments, the guide RNA includes a sequence complementary to a mammalian genome polynucleotide sequence. In some embodiments, the guide RNA includes a sequence complementary to a human genome polynucleotide sequence.

[0259] In some embodiments, the guide RNA is 30-250 nucleotides in length. In some embodiments, the guide RNA is more than 90 nucleotides in length. In some embodiments, the guide RNA is less than 245 nucleotides in length. In some embodiments, the guide RNA is 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, or more than 240 nucleotides in length. In some embodiments, the length of the guide RNA is about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 120, about 30 to about 140, about 30 to about 160, about 30 to about 180, about 30 to about 200, about 30 to about 220, about 30 to about 240, about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 100. One, about 50 to about 120, about 50 to about 140, about 50 to about 160, about 50 to about 180, about 50 to about 200, about 50 to about 220, about 50 to about 240, about 100 to about 120, about 100 to about 140, about 100 to about 160, about 100 to about 180, about 100 to about 200, about 100 to about 220, about 100 to about 240, about 160 to about 180, about 160 to about 200, about 160 to about 220 or about 160 to about 240 nucleotides.

[0260] In some embodiments, the left transposase recognition sequence includes a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 70% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 80% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 85% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 90% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 91% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 92% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 93% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 94% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 95% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 96% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 97% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78.In some embodiments, the left transposase recognition sequence includes a sequence having at least about 98% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 99% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having 100% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78.

[0261] In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 70% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 75% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 80% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 85% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 90% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 91% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 92% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 93% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 94% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 95% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 96% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93.In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 97% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 98% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 99% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having 100% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93.

[0262] In some embodiments, the type V Cas effector and Tn7 transposase complex are encoded by a polynucleotide sequence comprising less than about 20 kilobases, less than about 15 kilobases, less than about 10 kilobases, or less than about 5 kilobases.

[0263] In some embodiments, the type 2 V-effect includes a nuclear localization sequence (NLS). In some embodiments, the NLS is located at the N-terminus of the type 2 V-effect. In some embodiments, the NLS is located at the C-terminus of the type 2 V-effect. In some embodiments, the NLS is located at both the N-terminus and C-terminus of the type 2 V-effect.

[0264] In some embodiments, the NLS includes a sequence of any one of SEQ ID NO: 192-207 and 1354-1383 or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any one of SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS includes a sequence having at least about 80% identity with any one of SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 85% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 90% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 91% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 92% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 93% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 94% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 95% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 96% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 97% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 98% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 99% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having 100% identity with SEQ ID NO: 192-207 and 1354-1383.

[0265] Table 1: Exemplary NLS sequences

[0266]

[0267]

[0268] In some embodiments, the Cas effector complex further comprises the small prokaryotic ribosomal protein subunit S15. In some embodiments, the S15 fusion protein is encoded by a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 70% identity with any of SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 75% identity with any of SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 80% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 85% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 90% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 91% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 92% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 93% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 94% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 95% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 96% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 97% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 98% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 99% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having 100% identity with SEQ ID NO: 181-183.

[0269] In some embodiments, S15 includes a sequence having at least about 70% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 75% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 80% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 85% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 90% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 91% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 92% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 93% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 94% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 95% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 96% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 97% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 98% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 99% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having 100% identity with SEQ ID NO: 187-189.

[0270] In some embodiments, the Cas effector complex includes one or more adapters linking two types of V effectors, the small prokaryotic ribosomal protein subunit S15, a transposase, gRNA, or a combination thereof. In some embodiments, the adapter includes at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, or 400 amino acids. In some embodiments, the adapter includes at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides. In some embodiments, the connector is encoded by a sequence of SEQ ID NO: 186 or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 186. In some embodiments, the connector is encoded by SEQ ID NO: 186.

[0271] In some aspects, this disclosure provides an engineered nuclease system comprising: a nuclease including a RuvC domain, said nuclease being derived from an uncultured microorganism and being a type 2 VK Cas effector, said type 2 VK Cas effector having at least 80% identity with any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85 and 220; and an engineered guide RNA forming a complex with said nuclease and including a spacer sequence, said spacer sequence hybridizing with a target nucleic acid sequence, wherein said engineered guide polynucleotide includes a sequence having at least 80% identity with any one of SEQ ID NO: 754-944.

[0272] Fusion protein In some embodiments, this document describes a system for transposing a cargo nucleotide sequence into a target nucleic acid site, the system comprising a fusion protein or a nucleic acid encoding a fusion protein. In some embodiments, the fusion protein or the nucleic acid encoding a fusion protein comprises two types of V effectors, a small prokaryotic ribosomal protein subunit S15, a transposase, gRNA, or a combination thereof. In some embodiments, the fusion protein comprises one or more transposases.

[0273] In some embodiments, the nuclear localization sequence (NLS) is fused with two types of V-effects. In some embodiments, the NLS is fused at the N-terminus of the two types of V-effects. In some embodiments, the NLS is fused at the C-terminus of the two types of V-effects. In some embodiments, the NLS is fused at both the N-terminus and C-terminus of the two types of V-effects.

[0274] In some embodiments, the NLS includes a sequence of any one of SEQ ID NO: 192-207 and 1354-1383 or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any one of SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS includes a sequence having at least about 80% identity with any one of SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 85% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 90% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 91% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 92% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 93% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 94% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 95% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 96% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 97% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 98% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having at least about 99% identity with SEQ ID NO: 192-207 and 1354-1383. In some embodiments, the NLS comprises sequences having 100% identity with SEQ ID NO: 192-207 and 1354-1383.

[0275] In some embodiments, the fusion protein or the nucleic acid encoding the fusion protein comprises a fusion of S15 and a nuclear localization sequence (NLS). In some embodiments, the NLS is fused at the N-terminus of S15. In some embodiments, the NLS is fused at the C-terminus of S15. In some embodiments, the NLS is fused at both the N-terminus and C-terminus of S15.

[0276] In some embodiments, the S15 fusion protein further includes a cleavable peptide. In some embodiments, the peptide is a 2A peptide.

[0277] In some embodiments, the S15 fusion protein is encoded by a sequence having at least 80% sequence identity with any of SEQ ID NO: 181-183. In some embodiments, the S15 fusion protein is encoded by a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 181-183. In some embodiments, the Cas effector complex further comprises the small prokaryotic ribosomal protein subunit S15. In some embodiments, the S15 fusion protein is encoded by a sequence having at least 80% sequence identity with any of SEQ ID NO: 181-183. In some embodiments, the S15 fusion protein is encoded by a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 70% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 75% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 80% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 85% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 90% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 91% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 92% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 93% identity with SEQ ID NO: 181-183.In some embodiments, S15 is encoded by a sequence having at least about 94% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 95% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 96% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 97% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 98% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having at least about 99% identity with SEQ ID NO: 181-183. In some embodiments, S15 is encoded by a sequence having 100% identity with SEQ ID NO: 181-183.

[0278] In some embodiments, the S15 fusion protein comprises a sequence having at least about 70% sequence identity with any of SEQ ID NO: 187-189. In some embodiments, the S15 fusion protein has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 187-189. In some embodiments, S15 comprises a sequence having at least about 75% identity with any of SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 80% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 85% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 90% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 91% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 92% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 93% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 94% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 95% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 96% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 97% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 98% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having at least about 99% identity with SEQ ID NO: 187-189. In some embodiments, S15 includes a sequence having 100% identity with SEQ ID NO: 187-189.

[0279] In some embodiments, the NLS is fused with a transposase. In some embodiments, the transposase is TnsB, TnsC, or TniQ. In some embodiments, the transposase is TnsB. In some embodiments, the transposase is TnsC. In some embodiments, the transposase is TniQ. In some embodiments, the NLS is fused at the N-terminus of the transposase. In some embodiments, the NLS is fused at the C-terminus of the transposase. In some embodiments, the NLS is fused at both the N-terminus and C-terminus of the transposase.

[0280] In some embodiments, the fusion protein or the nucleic acid encoding the fusion protein includes the gRNA (e.g., dual gRNA or single gRNA) described herein.

[0281] In some embodiments, type V effectors, small prokaryotic ribosomal protein subunit S15, transposases, gRNA, or fusion proteins include tags. In some embodiments, the tag is an affinity tag. In some embodiments, the tag is a polypeptide or polynucleotide. Exemplary affinity tags include, but are not limited to, His tags, Flag tags, Myc tags, MBP tags, and GST tags.

[0282] In some embodiments, type V effectors, small prokaryotic ribosomal protein subunit S15, transposases, or fusion proteins include protease cleavage sites. Exemplary protease cleavage sites include, but are not limited to, TEV sites, C3 sites, factor Xa sites, and enterokinase sites.

[0283] cell In some embodiments, this document describes a cell that includes the system described herein.

[0284] In some embodiments, the cells are eukaryotic cells (e.g., plant cells, animal cells, protist cells, or fungal cells); mammalian cells (Chinese hamster ovary (CHO) cells, young hamster kidney (BHK) cells, human embryonic kidney (HEK) cells, mouse myeloma (NSO) cells, or human retinal cells); immortalized cells (e.g., HeLa cells, COS cells, HEK-293T cells, MDCK cells, 3T3 cells, PC12 cells, Huh7 cells, HepG2 cells, K562 cells, N2a cells, or SY5Y cells); insect cells (e.g., fall armyworm cells). Spodoptera frugiperda ) cells, Powdery noctuid moth ( Trichoplusia ni ) cells, Drosophila melanogaster ( Drosophila melanogaster ) cells, S2 cells or tobacco shoot moth ( Heliothis virescens ) cells); yeast cells (e.g., Saccharomyces cerevisiae ( Saccharomyces cerevisiaeThe cells can be eukaryotic cells, cryptococcal cells, or Candida cells; plant cells (e.g., parenchyma cells, collenchyma cells, or sclerenchyma cells); fungal cells (e.g., Saccharomyces cerevisiae cells, Cryptococcus cells, or Candida cells); or prokaryotic cells (e.g., Escherichia coli cells, Streptococcus cells, Streptomyces soil bacteria cells, or archaea cells). In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are immortalized cells. In some embodiments, the cells are insect cells. In some embodiments, the cells are yeast cells. In some embodiments, the cells are plant cells. In some embodiments, the cells are fungal cells. In some embodiments, the cells are prokaryotic cells.

[0285] In some embodiments, the cells are A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof.

[0286] Delivery and carrier In some embodiments, this document discloses nucleic acid sequences encoding the MG64 system or the gene editing system disclosed herein, which includes two types of V-effectors, a small prokaryotic ribosomal protein subunit S15, a transposase, gRNA, and a fusion protein.

[0287] In some embodiments, the nucleic acid encoding the MG64 system is DNA, such as linear DNA, plasmid DNA, or microcircular DNA. In some embodiments, the nucleic acid encoding the MG64 system is RNA, such as mRNA.

[0288] In some embodiments, the nucleic acid encoding the MG64 system is delivered by a nucleic acid-based vector. In some embodiments, the nucleic acid-based vector is a plasmid (e.g., a circular DNA molecule that can autonomously replicate within a cell), a phage (e.g., a pWE or sCos vector), an artificial chromosome, a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a P1-derived artificial chromosome (PAC), a phage particle, a phage derivative, a rod particle, or a virus. In some embodiments, the nucleic acid-based vector is selected from the list of the following: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEF1a-mCherry-N1 vector, pEF1a-tdTomato vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP tag(m), pSF-CMV-PURO-NH2-CMYC, pSF-OXB20-BetaGal, pSF-OXB20-Fluc, pSF-OXB20, pSF-Tac, pRI 101-AN DNA, pCambia2301, pTYB21, pKLAC2, pAc5.1 / V5-His A and pDEST8.

[0289] In some embodiments, the nucleic acid-based vector includes a promoter. In some embodiments, the promoter is selected from the group consisting of mini-promoters, inducible promoters, constitutive promoters, and derivatives thereof. In some embodiments, the promoter is selected from the group consisting of CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p19, p40, synaptic protein, CaMKII, GRK1, and derivatives thereof. In some embodiments, the promoter is the U6 promoter. In some embodiments, the promoter is the CAG promoter. In some embodiments, the promoter is encoded by a sequence of any of SEQ ID NO: 190-191 or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 190-191.

[0290] In some embodiments, the nucleic acid-based vector is a virus. In some embodiments, the virus is an alpha virus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the virus is an alpha virus. In some embodiments, the virus is a parvovirus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is AAV. In some embodiments, the virus is a baculovirus. In some embodiments, the virus is dengue virus. In some embodiments, the virus is a lentivirus. In some embodiments, the virus is a herpesvirus. In some embodiments, the virus is a poxvirus. In some embodiments, the virus is an anellovirus. In some embodiments, the virus is an anellovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is a retrovirus.

[0291] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, or derivatives thereof. In some embodiments, the herpesvirus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.

[0292] In some embodiments, the virus is AAV1 or a derivative thereof. In some embodiments, the virus is AAV2 or a derivative thereof. In some embodiments, the virus is AAV3 or a derivative thereof. In some embodiments, the virus is AAV4 or a derivative thereof. In some embodiments, the virus is AAV5 or a derivative thereof. In some embodiments, the virus is AAV6 or a derivative thereof. In some embodiments, the virus is AAV7 or a derivative thereof. In some embodiments, the virus is AAV8 or a derivative thereof. In some embodiments, the virus is AAV9 or a derivative thereof. In some embodiments, the virus is AAV10 or a derivative thereof. In some embodiments, the virus is AAV11 or a derivative thereof. In some embodiments, the virus is AAV12 or a derivative thereof. In some embodiments, the virus is AAV13 or a derivative thereof. In some embodiments, the virus is AAV14 or a derivative thereof. In some embodiments, the virus is AAV15 or a derivative thereof. In some embodiments, the virus is AAV16 or a derivative thereof. In some embodiments, the virus is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rh10 or a derivative thereof. In some embodiments, the virus is AAV-rh20 or a derivative thereof. In some embodiments, the virus is AAV-rh39 or a derivative thereof. In some embodiments, the virus is AAV-rh74 or a derivative thereof. In some embodiments, the virus is AAV-rhM4-1 or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the virus is AAV-Anc80 or a derivative thereof. In some embodiments, the virus is AAV-Anc80L65 or a derivative thereof. In some embodiments, the virus is AAV-7m8 or a derivative thereof. In some embodiments, the virus is AAV-PHP-B or a derivative thereof. In some embodiments, the virus is AAV-PHP-EB or a derivative thereof. In some embodiments, the virus is AAV-2.5 or a derivative thereof. In some embodiments, the virus is AAV-2tYF or a derivative thereof. In some embodiments, the virus is AAV-3B or a derivative thereof. In some embodiments, the virus is AAV-LK03 or a derivative thereof. In some embodiments, the virus is AAV-HSC1 or a derivative thereof. In some embodiments, the virus is AAV-HSC2 or a derivative thereof. In some embodiments, the virus is AAV-HSC3 or a derivative thereof. In some embodiments, the virus is AAV-HSC4 or a derivative thereof. In some embodiments, the virus is AAV-HSC5 or a derivative thereof. In some embodiments, the virus is AAV-HSC6 or a derivative thereof. In some embodiments, the virus is AAV-HSC7 or a derivative thereof. In some embodiments, the virus is AAV-HSC8 or a derivative thereof. In some embodiments, the virus is AAV-HSC9 or a derivative thereof.In some embodiments, the virus is AAV-HSC10 or a derivative thereof. In some embodiments, the virus is AAV-HSC11 or a derivative thereof. In some embodiments, the virus is AAV-HSC12 or a derivative thereof. In some embodiments, the virus is AAV-HSC13 or a derivative thereof. In some embodiments, the virus is AAV-HSC14 or a derivative thereof. In some embodiments, the virus is AAV-HSC15 or a derivative thereof. In some embodiments, the virus is AAV-TT or a derivative thereof. In some embodiments, the virus is AAV-DJ / 8 or a derivative thereof. In some embodiments, the virus is AAV-Myo or a derivative thereof. In some embodiments, the virus is AAV-NP40 or a derivative thereof. In some embodiments, the virus is AAV-NP59 or a derivative thereof. In some embodiments, the virus is AAV-NP22 or a derivative thereof. In some embodiments, the virus is AAV-NP66 or a derivative thereof. In some embodiments, the virus is AAV-HSC16 or a derivative thereof.

[0293] In some embodiments, the virus is HSV-1 or a derivative thereof. In some embodiments, the virus is HSV-2 or a derivative thereof. In some embodiments, the virus is VZV or a derivative thereof. In some embodiments, the virus is EBV or a derivative thereof. In some embodiments, the virus is CMV or a derivative thereof. In some embodiments, the virus is HHV-6 or a derivative thereof. In some embodiments, the virus is HHV-7 or a derivative thereof. In some embodiments, the virus is HHV-8 or a derivative thereof.

[0294] In some embodiments, the nucleic acid encoding the MG64 system is delivered by a non-nucleic acid-based delivery system (e.g., a non-viral delivery system). In some embodiments, the non-viral delivery system is a liposome. In some embodiments, the nucleic acid is associated with lipids. In some embodiments, the lipid-associated nucleic acid may be encapsulated within the aqueous interior of a liposome, dispersed in a lipid bilayer of a liposome, linked to a liposome via a linker molecule associated with both the liposome and the nucleic acid, captured in a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with lipids, incorporated with lipids, contained as a suspension in lipids, contained in or complexed with microparticles, or otherwise associated with lipids. In some embodiments, the nucleic acid is included in lipid nanoparticles (LNPs).

[0295] In some embodiments, the fusion protein or genome editing system is introduced into cells stably or transiently in any suitable manner. In some embodiments, the fusion protein or genome editing system is transfected into cells. In some embodiments, cells are transduced or transfected with a nucleic acid construct encoding the fusion protein or genome editing system. For example, cells are transduced (e.g., with a virus encoding the fusion protein or genome editing system), or transfected with nucleic acid encoding the fusion protein or genome editing system or a translated fusion protein or genome editing system (e.g., with a plasmid encoding the fusion protein or genome editing system). In some embodiments, the transduction is stable or transient. In some embodiments, for example, when the fusion protein or genome editing system includes a CRISPR nuclease, cells expressing the fusion protein or genome editing system or containing the fusion protein or genome editing system are transduced or transfected with one or more gRNA molecules. In some embodiments, plasmids expressing the fusion protein or genome editing system are introduced into cells via electroporation, transient (e.g., lipid transfection), and stable genome integration (e.g., piggybac), as well as viral transduction (e.g., lentivirus or AAV) or other methods known to those skilled in the art. In some embodiments, the gene editing system is introduced into cells as one or more peptides. In some embodiments, delivery is achieved by using an RNP complex. Methods for delivering peptides and / or RNPs to cells are known in the art, such as by electroporation or by cell extrusion.

[0296] Exemplary methods for nucleic acid delivery include lipid transfection, nuclear transfection, electroporation, stable genome integration (e.g., piggybac), microinjection, gene guns (biolistics), virions, liposomes, immunoliposomes, polycationic or lipid nucleic acid conjugates, naked DNA, artificial viral particles, and drug-enhanced DNA uptake. Lipid transfection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipid transfection reagents are commercially available (e.g., Transfectam™, Lipofectin™, and SF cell line 4D-nuclear transfectant X Kit™ (Lonza)). Cationic and neutral lipids suitable for highly efficient receptor recognition of polynucleotides for lipid transfection comprise cationic and neutral lipids as described in WO 91 / 17424 and WO 91 / 16024. In some embodiments, delivery is made to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). In some embodiments, nucleic acids are contained in liposomes or nanoparticles that specifically target host cells.

[0297] Other methods for delivering nucleic acids into cells are known to those skilled in the art. See, for example, US 2003 / 0087817.

[0298] In some embodiments, this disclosure provides a cell comprising the vector or nucleic acid described herein. In some embodiments, the cell expresses a gene editing system or a portion thereof. In some embodiments, the cell is a human cell. In some embodiments, the cell is an ex vivo edited genome. In some embodiments, the cell is an in vivo edited genome.

[0299] Methods for transposition This disclosure provides methods for transposing cargo nucleotide sequences into target nucleic acid sites. In some embodiments, the methods include expressing the system described herein in cells or introducing the system described herein into cells. In some embodiments, the methods include contacting cells with the system described herein.

[0300] In some embodiments, the method includes contacting a double-stranded nucleic acid including a cargo nucleotide sequence with a Cas effector complex, the Cas effector complex including two types V Cas effectors and at least one engineered guide polynucleotide configured to hybridize with a target nucleotide sequence. In some embodiments, the method includes contacting a double-stranded nucleic acid including a cargo nucleotide sequence with a Tn7 transposase complex configured to bind to the Cas effector complex, wherein the Tn7 transposase complex includes a TnsB subunit. In some embodiments, the method includes contacting a double-stranded nucleic acid including a cargo nucleotide sequence with a double-stranded target nucleic acid including a target nucleic acid site.

[0301] In some embodiments, the cargo nucleotide sequence is flanked by a left-hand transposase recognition sequence. In some embodiments, the cargo nucleotide sequence is flanked by a right-hand transposase recognition sequence. In some embodiments, the cargo nucleotide sequence is flanked by both a left-hand transposase recognition sequence and a right-hand transposase recognition sequence. In some embodiments, the method further includes a PAM sequence compatible with the Cas effector complex adjacent to the target nucleic acid site. In some embodiments, the PAM sequence is located at the 3' position of the target nucleic acid site.

[0302] In some embodiments, engineered guide polynucleotides are configured to bind to type 2 V Cas effectors. In some embodiments, type 2 V Cas effectors include polypeptides comprising sequences having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 70% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 75% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 80% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 85% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 90% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 91% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 92% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 93% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, type 2 V-type Cas effectors include polypeptides comprising sequences having at least about 94% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85 and 220.In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 95% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 96% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 97% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the type 2 V Cas effector comprises a polypeptide containing a sequence having at least about 98% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, type 2 V Cas effectors include polypeptides comprising sequences having at least about 99% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, type 2 V Cas effectors include polypeptides comprising sequences having 100% identity with SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220.

[0303] In some embodiments, the TnsB subunit comprises a polypeptide having a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 70% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 75% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 80% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 85% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 90% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 91% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 92% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 93% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 94% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 95% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 96% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 97% identity with SEQ ID NO: 2, 13, 17, and 65.In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 98% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having at least about 99% identity with SEQ ID NO: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide containing a sequence having 100% identity with SEQ ID NO: 2, 13, 17, and 65.

[0304] In some embodiments, the Tn7 transposase complex comprises at least one polypeptide (e.g., at least 1, 2, 3, 4, 5, 6 or more polypeptides) comprising a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity with any of SEQ ID NO: 3-4, 14-15, 18-19, 66-67 and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 70% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 75% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 80% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 85% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 90% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 91% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 92% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 93% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111.In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 94% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 95% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 96% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide containing a sequence having at least about 97% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide having a sequence having at least about 98% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide having a sequence having at least about 99% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises a polypeptide having a sequence having 100% identity with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111.

[0305] In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently comprising a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently comprising a sequence having at least 70% sequence identity with any one of SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently comprising a sequence having at least 75% sequence identity with any one of SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 85% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 90% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 91% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111.In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 92% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 93% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 94% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 95% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 96% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence identity of at least about 97% with SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence with at least about 98% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence with at least about 99% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide independently having a sequence with 100% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111.

[0306] In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 70% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 75% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 80% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 85% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 90% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 91% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 92% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122 and 222.In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 93% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 94% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 95% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 96% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 97% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 98% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least about 99% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having 100% identity with SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122, and 222.

[0307] In some embodiments, the engineered guide polynucleotide is a guide RNA and includes a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence identical to any one of SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 70% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 75% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 80% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 85% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 90% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising sequences having at least about 91% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140 and 754-944.In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 92% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 93% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 94% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 95% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 96% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 97% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 98% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having at least about 99% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944. In some embodiments, the engineered guide polynucleotide is a guide RNA comprising a sequence having 100% identity with SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140, and 754-944.

[0308] In some embodiments, the left transposase recognition sequence includes a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 70% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 80% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 85% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 90% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 91% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 92% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 93% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 94% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 95% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 96% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 97% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78.In some embodiments, the left transposase recognition sequence includes a sequence having at least about 98% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having at least about 99% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78. In some embodiments, the left transposase recognition sequence includes a sequence having 100% identity with SEQ ID NO: 9, 11, 36-38, 76, and 78.

[0309] In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 70% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 75% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 80% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 85% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 90% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 91% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 92% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 93% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 94% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 95% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 96% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93.In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 97% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 98% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having at least about 99% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the right-sided transposase recognition sequence includes a sequence having 100% identity with SEQ ID NO: 8, 10, 39-44, 77, 79, and 93.

[0310] In some embodiments, the type V Cas effector and Tn7 transposase complex are encoded by a polynucleotide sequence comprising less than about 20 kilobases, less than about 15 kilobases, less than about 10 kilobases, or less than about 5 kilobases.

[0311] use The systems disclosed herein can be used for a variety of applications, such as nucleic acid editing (e.g., gene editing) or binding to nucleic acid molecules (e.g., sequence-specific binding). Such systems can be used, for example, to remedy (e.g., remove or replace) genetic mutations that may cause disease in a subject; to inactivate genes to determine their function in cells; as diagnostic tools for detecting pathogenic genetic elements (e.g., by cleaving retroviral RNA or amplified DNA sequences encoding pathogenic mutations); as inactivating enzymes bound to probes to target and detect specific nucleotide sequences (e.g., sequences encoding bacterial antibiotic resistance); to inactivate viruses or prevent them from infecting host cells by targeting viral genomes; to engineer organisms by adding genes or modifying metabolic pathways to produce valuable small molecules, macromolecules, or secondary metabolites; to establish gene-driven elements for evolutionary selection; and / or as biosensors to detect interference from foreign small molecules and nucleotides in cells.

[0312] Reagent test kit In some embodiments, this disclosure provides a kit comprising one or more nucleic acid constructs encoding various components of the fusion protein or genome editing system described herein, for example, comprising nucleotide sequences encoding components of a fusion protein or genome editing system capable of modifying a target DNA sequence. In some embodiments, the nucleotide sequences include heterologous promoters driving the expression of components of an RNA genome editing system.

[0313] In some embodiments, gene editing systems comprising two types of V-effectors, small prokaryotic ribosomal protein subunit S15, transposases, gRNAs, or fusion proteins, or any combinations thereof disclosed herein, are assembled into a pharmaceutical, diagnostic, or research kit to facilitate their use in therapeutic, diagnostic, or research applications. The kit may contain one or more containers housing any of the vectors disclosed herein and instructions for use.

[0314] The kit may be designed to facilitate researchers' use of the methods described herein and may take many forms. Each composition of the kit may be provided, where applicable, in liquid form (e.g., in solution) or in solid form (e.g., dry powder). In some cases, some compositions may be constructible or otherwise processable (e.g., to form an active form), for example by adding a suitable solvent or other species (e.g., water or cell culture medium). These compositions may or may not be provided with the kit. As used herein, “instructions” may define the instructions and / or promoted composition and generally refer to written instructions on or associated with the packaging of this disclosure. Instructions may also include any oral or electronic instructions provided in any way that makes it clear to the user that the instructions will be related to the kit, e.g., audiovisual (e.g., videotape, DVD, etc.), internet and / or network-based communications, etc. In some embodiments, written instructions are in the form prescribed by a government agency regulating the manufacture, use or sale of pharmaceuticals or biological products, and may also reflect the agency’s authorization for animal administration in relation to manufacture, use or sale.

[0315] Example The following examples are given for the purpose of illustrating various embodiments of this disclosure and are not intended to limit this disclosure in any way. These examples and the methods described herein represent exemplary preferred embodiments and are not intended to limit the scope of this disclosure. Variations and other uses covered within the spirit of this disclosure as defined by the claims will be apparent to those skilled in the art.

[0316] Example 1 - (General Solution) PAM Sequence Identification / Confirmation of the System Described in This Paper The putative endonuclease was expressed in an *E. coli* lysate-based expression system. The PAM sequence was determined by sequencing a plasmid containing a randomly generated potential PAM sequence that could be cleaved by the putative endonuclease. In this system, under the control of the T7 promoter, an *E. coli* codon-optimized nucleotide sequence encoding the putative endonuclease was transcribed and translated in vitro from a PCR fragment. In the same reaction, a second PCR fragment with a minimal CRISPR array consisting of the T7 promoter and subsequent repeat-spacer-repeat sequences was transcribed. Successful expression of the endonuclease and repeat-spacer-repeat sequences in the *E. coli* lysate-based expression system, followed by CRISPR array processing, provided an active in vitro CRISPR endonuclease complex.

[0317] A library of target plasmids containing a spacer sequence matching the spacer sequence preceding the 8N mixed bases (potential PAM sequence) in the minimal array is incubated together with the reaction output of the expression system. After 1–3 hours, the reaction is stopped, and the DNA is recovered using a DNA cleaning kit. The blunt ends of the adaptor sequence are ligated to DNA containing an active PAM sequence that has been cleaved by a nuclease, while uncut DNA cannot be approached for ligation. The DNA segment containing the active PAM sequence is then amplified by PCR using primers specific to the library and adaptor sequences. The PCR amplification products are resolved on a gel to identify the amplicons corresponding to the cleavage events. The amplified segments from the cleavage reaction are also used as templates for preparing NGS libraries or as substrates for Sanger sequencing. The resulting library, a subset of the initial 8N library, is sequenced, revealing a sequence with PAM activity compatible with the CRISPR complex. For PAM tests using the treated RNA construct, the same procedure was repeated, except that the minimal CRISPR array template was omitted, except that the in vitro transcribed RNA was added along with the plasmid library.

[0318] Analysis of the Cas effector and the intergenic region surrounding the CRISPR array identified potential anti-repetition sequences corresponding to the tracrRNA duplex sequence. The tracrRNA and crRNA repeat sequences were folded and pruned, and a GAAA tetraloop sequence was added to maintain the stem-loop region of the crRNA-tracrRNA complex.

[0319] Example 2A - In vitro targeted integrase activity Integrase activity was determined using previously identified PAMs, but alternatively, it could be determined using PAM library substrates, where efficiency was reduced. One arrangement of components for in vitro testing involved three plasmids in addition to containing donor sequences: (1) an expression plasmid with one (or more) effectors under the T7 promoter; (2) an expression plasmid with a transposase gene under the T7 promoter; sgRNA or crRNA and tracrRNA; (3) a target plasmid containing a spacer site and an appropriate PAM; and (4) a donor plasmid containing the desired left-hand (LE) and right-hand (RE) DNA sequences for transposition around a cargo gene (e.g., a selection marker, such as the Tet resistance gene). The effector and transposase gene were expressed using an in vitro transcription / translation system (e.g., a system based on E. coli lysate or reticulocyte lysate). After expression, RNA, target DNA, and donor DNA were added and incubated to allow transposition. Transposition was detected by PCR at the junction of transposase sites, with one primer on the target DNA and one primer on the donor DNA. The resulting PCR product was sequenced by NGS to determine the exact insertion topology relative to the sgRNA / crRNA target site. The primers were positioned downstream to accommodate and detect various insertion sites. The primers were designed to detect integration in either orientation of the cargo and on either side of the spacer, as the integration orientation had not been previously recorded.

[0320] Integration efficiency was measured by quantitative PCR (qPCR) of the experimental output of target DNA and integrated cargo, and normalized relative to the amount of unmodified target DNA, which was also measured by qPCR.

[0321] This assay can be performed using purified protein fractions, rather than from lysate-based expression. In this case, the protein was expressed in *E. coli* protease-deficient B strain under the T7 inducible promoter, cells were lysed using sonication, and the His-labeled protein of interest was purified on an FPLC system using Ni-NTA affinity chromatography. Purity was determined using density determination of protein bands separated on SDS-PAGE and Coomassie-stained acrylamide gels. The protein was desalted in a storage buffer consisting of 50 mM Tris-HCl, 300 mM NaCl, 1 mM TCEP, and 5% glycerol; pH 7.5 (or another buffer as determined for maximum stability) and stored at -80°C. After purification, the effector and transposase were added to a reaction buffer containing sgRNA, target DNA, and donor DNA as described above, for example, supplemented with 26 mM HEPES (pH 7.5), 4.2 mM TRIS (pH 8), 50 µg / mL BSA, 2 mM ATP, 2.1 mM MTT, 0.05 mM EDTA, 0.2 mM MgCl2, 28 mM NaCl, 21 mM KCl, and 1.35% glycerol (final pH 7.5).

[0322] Example 2B - In vitro activity Targeted nuclease In situ Expression and protein sequence analysis indicated that some RNA-guided effectors are active nucleases. They contain predicted endonuclease-associated domains (matching RuvC and HNH_ endonuclease domains) and / or predicted HNH and RuvC catalytic residues.

[0323] Candidate activity was tested using an expression system based on E. coli lysates and in vitro transcribed RNA, with engineered single-guide RNA sequences. Successful cleavage of the library yielded an approximately 170 bp band in the gel.

[0324] DNA integration and transposition A transposon is predicted to be active when the genomic sequence encoding it contains one or more protein sequences with transposase and / or integrase functions at its left and right ends. As defined herein, a Tn7 transposon may include the catalytic transposase TnsB, but may also contain TnsA, TnsC, TnsD, TnsE, TniQ, and / or other transposases or integrases. The transposon ends include a predicted transposase binding site containing direct and / or inverted repeat sequences of 15 bp to 150 bp, flanked by the transposase protein and other “cargo” genes. Protein sequence analysis indicating that the transposase contains an integrase domain, a transposase domain, and / or transposase catalytic residues indicates that it is active (e.g., Figure 4A ).

[0325] Targeted DNA integration CRISPR-associated transposons (CASTs) are presumed to contain DNA and / or RNA targeting CRISPR nucleases or effectors, as well as proteins near the CRISPR array that predict transposase function. In some systems, the activity of the nuclease is predicted based on the presence of nuclease-associated catalytic domains and / or catalytic residues.

[0326] In some systems, predicted effectors share homology with recorded CRISPR effector proteins, but are inactive due to the absence of nuclease domains and / or catalytic residues. Predicted transposases are associated with effectors when the CRISPR locus (inactive CRISPR nuclease and array) and transposase proteins are located at the left and right ends of the predicted transposon. Figure 4A In this context, predictive effectors guide DNA integration to specific genomic locations based on guide RNAs.

[0327] CAST activity was tested using five types of components: (1) Cas effector proteins expressed by an in vitro expression system; (2) target DNA fragments or plasmids containing the target sequence and a PAM corresponding to the Cas enzyme; (3) donor DNA fragments containing DNA markers or fragments of the transposase system flanked by LE and RE; (4) any combination of transposase proteins expressed using an in vitro expression system; and (5) engineered in vitro transcribed single-guide RNA sequences. The activity of successfully transposable donor fragments was determined by PCR amplification of the donor-target junction.

[0328] Following the transposition reaction, PCR amplification of the junction site demonstrated proper donor-target formation, and the transposition reaction was sg-dependent. Figure 6PCR amplification in reactions #3 and #4 indicated two donor orientations relative to the target: one where LE was closer to PAM, and the other where RE was closer to PAM. Although both transposon orientations were observed, a preference for donor integration was present in the target where LE was closer to PAM, indicated by strong bands in reactions #4 and #5.

[0329] Sanger sequencing was performed on the oriented products. In integration occurring where the LE is closer to the PAM, the sequencing chromatogram signal showed significant attenuation in either the forward or reverse direction at the target / donor junction. This indicates that in products oriented closer to the PAM, integration occurs across a series of nucleotides, with the major product of the LE product closer to the PAM being a 61 bp integration from the PAM. Figure 7A Sequencing from the donor at the donor-target junction defines the composition of the basic outer boundaries of the LE and RE sequences. Figures 7A-7B Further investigation of the LE and RE domains could identify internal restrictions in the LE and RE sequences crucial for transposition. Sequencing of the RE on the LE product closer to PAM revealed a 3 bp repeat downstream of the donor RE. Figure 7B This is partly due to the Tn7 transposase integration event that cleaves and ligates the donor fragment at the staggered cleavage site. The 3 bp repeat is smaller than the expected 5 bp repeat from other Tn7 transposases.

[0330] Sanger sequencing of PCR amplification products on the 8N library of the target plasmid further elucidated the PAM preference of the MG64-1 effector at the 5' end of the spacer for nGTn / nGTt. Figure 7C NGS analysis of the PAM library target confirmed the nGTn motif specificity at the 5' end.

[0331] Example 3 - Predicting RNA Folding The predicted RNA folding of the active single RNA sequence was calculated at 37 °C using the Andronescu 2007 method. All hairpin loop secondary structures were individually deleted from the structure and iteratively compiled into smaller single guides. In a second method, the tracrRNA of MG64-1 was aligned with the recorded Vk-type tracrRNA, and the uniquely inserted region was mutated into a single guide, reducing it by a maximum of 57 bases. Figure 12A The predicted structure of MG64-1 sgRNA was depicted. Figure 12B The predicted structure of MG64-3 sgRNA was depicted. Figure 12C The predicted structure of MG64-5 sgRNA was depicted. The base colors correspond to the probability of base pairing, with red indicating high probability and blue indicating low probability.

[0332] Example 4 - Transposon End Verification via Gel Translocation TnsB binding at the transposon ends was tested by electrophoretic mobility shift assay (EMSA). In this case, potential LEs or REs were synthesized as DNA fragments (100–500 bp) and end-labeled with FAM via PCR using primers with FAM labels. TnsB protein was synthesized in an in vitro transcription / translation system. After synthesis, 1 µL of TnsB protein was added to 50 nM labeled REs or LEs in a 10 µL reaction in binding buffer (20 mM HEPES pH 7.5, 2.5 mM Tris pH 7.5, 10 mM NaCl, 0.0625 mM EDTA, 5 mM TCEP, 0.005% BSA, 1 µg / mL poly(dI-dC), and 5% glycerol). The binding was incubated at 30˚ for 40 min, followed by the addition of 2 µL of 6X loading buffer (60 mM KCl, 10 mM Tris pH 7.6, 50% glycerol). The binding reaction was isolated and visualized on a 5% TBE gel. The transposition of LE or RE in the presence of TnsB is attributed to successful binding and indicates transposase activity. Figure 24 ).

[0333] Example 5 - Integrase activity in Escherichia coli Because *E. coli* lacks the ability to effectively repair double-stranded DNA breaks in its genome, transformation of *E. coli* with agents capable of inducing double-strand breaks in the *E. coli* genome leads to cell death. Utilizing this phenomenon, endonuclease or effector-assisted integrase activity was tested in *E. coli* by recombinantly expressing endonucleases or effector-assisted integrase and guide RNA (e.g., as determined in Example 3), where the spacer / target and PAM sequences are integrated into its genomic DNA.

[0334] Then, engineered strains were transformed with plasmids containing nucleases or effectors with single-path guide RNAs, plasmids expressing integrase and helper genes, and plasmids containing temperature-sensitive origins of replication (OCRs) with selectable markers flanked by left-hand (LE) and right-hand (RE) transposon motifs for integration. Transformants inducing these gene expression were then screened by selecting plasmids that replicated at a controlled temperature to transfer the markers to genomic targets, and PCR was used to confirm the integration of the markers into the genome.

[0335] Off-target integration was screened using an unbiased method. In short, purified gDNA was fragmented using Tn5 transposase or cleavage, and then the DNA of interest was amplified by PCR using primers specific to the ligation adaptor and selectable markers. Amplicon was then prepared for NGS sequencing. Analysis of the resulting sequences involved trimming transposon sequences and mapping side sequences to the genome to determine insertion sites and off-target insertion rates.

[0336] Example 6 - Colony PCR Screening for Transposase Activity To test the activity of nucleases or effector-assisted integrase in bacterial cells, strain MGB0032 was constructed from BL21(DE3) *E. coli* cells engineered to contain a target specific to MG64_1 and the corresponding PAM sequence. The MGB0032 *E. coli* cells were then transformed with pJL56 (a plasmid expressing the MG64_1 effector and helper kit, ampicillin resistant) and pTCM64_1sg (a chloramphenicol resistant plasmid expressing a single guide RNA sequence of the engineered target of interest driven by the T7 promoter).

[0337] MGB0032 cultures containing both plasmids were then grown to saturation, diluted at least 1:10 with an appropriate antibiotic, and incubated at 37°C until the OD was approximately 1. Cells from this growth stage were made electrocompetent and transformed with streamlined 64_1 pDonor, a plasmid carrying a tetracycline resistance marker with left- and right-handed (LE) transposon motifs for integration. Electroporated cells were then recovered from LB medium for 2 hours with or without a final concentration of 100 µM IPTG, plated on LB agar-ampicillin-chloramphenicol-tetracycline, and incubated at 37°C for 4 days. Each resulting CFU mixed in water was sampled using a sterile toothpick. Q5 high-fidelity PCR master mix (New England Biolabs) and primers LA155 (5'-GCTCTTCCGATCTNNNNNGATGAGCGCATTGTTAGATTTCAT-3' (SEQ ID NO: 1256)) and oJL50 (5'-AAACCGACATCGCAGGCTTC-3' (SEQ ID NO: 1257)) were added to this solution. These primers were side-joined to the predicted insertion junction. The predicted product size was 609 bp. The PCR product of DNA amplification was visualized on a 2% agarose gel. Sanger sequencing of the PCR product confirmed the transposition event.

[0338] Example 7 - Intracellular Expression / In vitro Assay To test the function of the NLS construct in a physiologically relevant setting, constructs containing clones of the active NLS-labeled CAST component were integrated into K562 cells using lentiviral transduction. Briefly, the construct cloned into a lentiviral transfer plasmid was transfected into 293T cells using an encapsulation and packaging plasmid, and the virus containing supernatant was collected from the culture medium after 72 hours of incubation. The virus-containing medium was then incubated with the K562 cell line and 8 µg / mL polybrene for 72 hours. Transfected cells were then selected for bulk integration using 1 µg / mL puromycin over 4 days. At the end of 4 days, the selected cell lines were collected, and differential lysis was performed targeting the nuclear and cytoplasmic fractions. The transposability of subsequent fractions with a set of complementary in vitro expressed components was then tested.

[0339] 10 million cells were centrifuged and washed once with 1x PBS pH 7.4. The supernatant was completely aspirated into the cell pellet and rapidly frozen at -80°C for 16 hours. After thawing on ice, the cell pellet size was measured by mass, and proteins in the cell fraction were naturally extracted using an appropriate extraction volume of cell fraction and nuclear extraction reagent (NE-PER). Briefly, the cytoplasmic extraction reagent was used at a 1:10 ratio of cell mass to extraction reagent volume. The cell suspension was mixed by vortexing and lysed with a non-ionic detergent. The cells were then centrifuged at 16,000 xg for 5 minutes at 4°C. The cytoplasmic extraction supernatant was then decanted and stored for in vitro testing. The nuclear extraction reagent was then added to the nuclear extraction reagent at a 1:2 ratio of the original cell mass and incubated on ice by intermittent vortexing for 1 hour. The nuclear suspension was then centrifuged at 16,000 xg for 10 minutes at 4°C, and the supernatant nuclear extract was decanted and in vitro transposition activity was tested. For each condition, in vitro transposition reactions were performed using 4 µL of each cell and nuclear extract, with a set of complementary in vitro expressed proteins, donor DNA, pTarget, and buffer. Evidence of transposition activity was determined by PCR amplification of the donor-target junction.

[0340] Example 8 - Activity (Prediction) in Mammalian Cells To demonstrate targeting and cleavage activity in mammalian cells, nuclear localization sequences were fused to the C-terminus of each of the nuclease or effector protein and integrase protein, and the fusion protein was purified. Single-guide RNA targeting the locus of interest in the genome was synthesized and incubated with the nuclease / effector protein to form a ribonucleoprotein complex. Cells were transfected with plasmids containing a selectable neomycin resistance marker (NeoR) or fluorescent markers flanked by left-hand (LE) and right-hand (RE) motifs, recovered for 4–6 hours, and subsequently electroporated with nuclease RNP and integrase protein. Quantification of plasmid integration into the genome was performed by counting G418 resistance colonies or by fluorescence-activated cell cytometry. Genomic DNA was extracted 72 hours after electroporation and used for NGS library preparation. Off-target frequencies were determined by fragmenting the genome and preparing amplicon and flanked DNA of transposon markers for NGS library preparation. At least 40 different target sites were selected to test the activity of each target system.

[0341] Example 9 - Targeting nuclease activity In situ expression and protein sequence analysis revealed that some RNA-guided effectors are active nucleases. These contain predicted endonuclease-associated domains (matching RuvC and HNH_ endonuclease domains) and predicted HNH and RuvC catalytic residues (…). Figure 4A ).

[0342] Candidate activity was tested using engineered single-guide RNA sequences via an in vitro expression system and in vitro transcribed RNA. Successful cleavage of the library of active proteins produced approximately 170 bp bands in the gel.

[0343] Example 10 - Identification of Transposons A transposon is predicted to be active when it contains one or more protein sequences with transposase and / or integrase function between its left and right ends. As defined herein, a Tn7 transposon may include the catalytic transposase TnsB, but may also contain TnsA, TnsC, TnsD, TnsE, TniQ, and / or other transposases or integrases. The transposon ends include a predicted transposase binding site containing direct and / or inverted repeat sequences of 15 bp to 150 bp, flanked by the transposase protein and other “cargo” genes. Protein sequence analysis indicating that the transposase contains an integrase domain, a transposase domain, and / or transposase catalytic residues indicates that it is active (e.g., Figure 4A and Figure 5 (Small image A).

[0344] Example 11 - Identification of CRISPR-related transposons CRISPR-associated transposons (CASTs) are presumed to contain DNA and / or RNA targeting CRISPR effectors, as well as proteins with predicted transposase function in the vicinity of the CRISPR array. In some systems, effectors are predicted to have nuclease activity based on the presence of nuclease-associated catalytic domains and / or catalytic residues (e.g., Figure 4A When the CRISPR locus (CRISPR nuclease and array) and transposase protein are located between the left and right ends of the predicted transposon, the predicted transposon is associated with the active nuclease (e.g., Figure 4B -4C). In this case, predictive effectors guide DNA integration to a specific genomic location based on guide RNA.

[0345] In some systems, predicted effectors share homology with recorded CRISPR effector proteins, but are inactive due to the absence of endonuclease domains and / or catalytic residues. Figure 5 (See small figure A). When the CRISPR locus (inactive CRISPR nuclease and array) and transposase protein are located within the left and right ends of the predicted transposon, the predicted transposon is associated with the effector. Figure 5 (Small pictures A and B).

[0346] Example 12 - CAST Identification CRISPR-associated transposons (CASTs) are systems that include transposons that have evolved to interact with the CRISPR system to facilitate the targeted integration of DNA cargo.

[0347] CAST is a genomic sequence encoding one or more protein sequences relating to DNA transposition within the characteristic left and right ends of a transposon. As defined herein, the Tn7 transposon may include the catalytic transposase TnsB, but may also contain the catalytic transposase TnsA, the loading protein TnsC or TniB, and the target recognition proteins TnsD, TnsE, TniQ, and / or other transposon-related components. The transposon ends include a predicted transposase binding site containing direct and / or inverted repeat sequences of 15 bp to 150 bp, flanking the transposon mechanism and other "cargo" genes.

[0348] Additionally, CAST encodes DNA and / or RNA targeting CRISPR nucleases or effectors near the CRISPR array. In some systems, the predicted effector is an active nuclease based on the presence of endonuclease-associated catalytic domains and / or catalytic residues. In other systems, the predicted effector has sequence similarity to the recorded CRISPR effector protein but is inactive due to the absence of endonuclease domains and / or catalytic residues. The predicted transposon is associated with the effector when CRISPR loci and transposon-associated proteins are located within the left and right ends of the predicted transposon. In this case, the predicted effector guides DNA integration to a specific genomic location based on guide RNA.

[0349] Example 13 - Class 2 Cas12K CAST The Cas12k CAST system encodes nuclease-deficient CRISPR Cas12k effectors, CRISPR arrays, tracrRNA, and Tn7-like transposons. The Cas12k effectors are phylogenetically distinct, and several characteristics have been confirmed to confirm their association with CAST. Figure 8 For example, the left end of the transposon is identified downstream of the MG64-3 CRISPR locus, as shown by the terminal inverted repeat sequence and the self-matching spacer sequence. Figure 11A The Cas12k CAST CRISPR repeat sequence (crRNA) contains the conserved motif 5'-GNNGGNNTGAAAG-3' ( Figure 9 Aligning short repeat-anti-repetition sequences (RARs) within the crRNA motif with different regions of tracrRNA. Figure 9 (and Figure 10), and the RAR motif appears to define the start and end of the tracrRNA (e.g., for MG64-1, the 5' end of the tracrRNA contains RAR1 (TTTC), and the 3' end contains RAR2 (CCNNC), Figure 10A ).

[0350] Example 14 - Transposon Prediction Transposon ends are estimated from intergenic regions based on lateral effectors and transposon mechanisms. For example, for Cas12k CAST, the intergenic region located directly upstream of TnsB and downstream of the CRISPR locus is predicted to contain the left and right ends (LE and RE) of the Tn7 transposon.

[0351] Predict approximately 12 bp direct and inverted repeat sequences (DR / IR) with at most two mismatches on the contiguous group. Additionally, a dot plot algorithm is used to locate short (approximately 10-20 bp) DR / IRs flanking CAST transposons. Matching DR / IRs encoding transposon binding sites are predicted in the intergenic regions of the flanking CAST effector and transposon genes. LE and RE sequences extracted from the intergenic regions encoding the putative transposon binding sites are aligned to define transposon end boundaries. The putative transposon LE and RE ends are regions that: a) are located within 400 bp upstream and downstream of the coding genes of the first and last predicted transposons; b) share multiple short inverted repeat sequences; and c) share >65% of the nucleotide IDs.

[0352] Example 15 - One-Way Design Analysis of the intergenic regions surrounding the Cas effector and CRISPR array identified potential anti-repetition sequences and a conserved “CYCC(n6)GGRG” stem-loop structure adjacent to an anti-repetition sequence corresponding to a tracrRNA duplex sequence. Figure 11B TracrRNA and crRNA repeat sequences were folded and pruned, and GAAA quadruple loop sequences were added to maintain the stem-loop region of the crRNA-tracrRNA complementary sequence.

[0353] Example 16 - In vitro integration activity using targeted nucleases In situ expression and protein sequence analysis indicated that some RNA-guided effectors were active nucleases. These contained predicted endonuclease-associated domains (matching RuvC and HNH_ endonuclease domains) and / or predicted HNH and RuvC catalytic residues. Candidate activities were tested using engineered single-guide RNA sequences with an in vitro expression system and in vitro transcribed RNA. Active proteins that successfully cleaved the library produced approximately 170 bp bands in the gel.

[0354] Example 17 - Programmable DNA Integration CAST activity was tested using five types of components: (1) Cas effector proteins expressed in an in vitro expression system (SEQ ID NO: 1); (2) target DNA fragments or plasmids containing the target sequence and the corresponding PAM for the Cas enzyme (SEQ ID NO: 31); (3) donor DNA fragments containing DNA markers or fragments of the transposase system flanked by LE and RE in the DNA fragment or plasmid (SEQ ID NO: 8-11); (4) any combination of transposase proteins expressed using an in vitro expression system (SEQ ID NO: 2-4); and (5) engineered in vitro transcribed single-guide RNA sequences (SEQ ID NO: 5). The activity of successfully transposable donor fragments was determined by PCR amplification of the donor-target junction.

[0355] Following the transposition reaction, PCR amplification of the junction site showed appropriate donor-target formation, and the transposition reaction was sg-dependent. Figure 9 PCR amplification in reactions #3 and #4 indicated two donor orientations relative to the target: one where LE was closer to PAM, and the other where RE was closer to PAM. Although both transposon orientations were observed, a donor integration preference appeared in the target where LE was closer to PAM, as indicated by the strong bands present in reactions #4 and #5.

[0356] Sanger sequencing was performed on the oriented products. In integration occurring where the LE is closer to the PAM, the sequencing chromatogram signal showed significant attenuation in either the forward or reverse direction at the target / donor junction. This indicates that in products oriented closer to the PAM, integration occurs across a series of nucleotides, with the major product of the LE product closer to the PAM being a 61 bp integration from the PAM. Figure 10A Sequencing from the donor at the donor-target junction defines the composition of the basic outer boundaries of the LE and RE sequences. Figure 10A , Figure 10B Sequencing of the RE on the LE product closer to PAM revealed a 3bp repeat downstream of the donor RE. Figure 10B This is partly due to the Tn7 transposase integration event that cleaves and ligates the donor fragment at the staggered cleavage site. The 3 bp repeat is smaller than the expected 5 bp repeat from other Tn7 transposases.

[0357] Sanger sequencing of PCR amplification products on the 8N library of the target plasmid also indicated that the MG64-1 effector was a PAM preference for nGTn / nGTt at the 5' end of the spacer (Figure 10C). NGS analysis of the PAM library target confirmed the nGTn motif selectivity at the 5' end.

[0358] Further development of the single-guided assay confirmed the activity of MG64-1 with the novel sgRNA scaffold (Figure 13).

[0359] Example 18 - Confirm Integrated Window The PCR junctions of amplified PAM were indexed against the NGS library and sequenced. Amplicon sequences at a 60 bp integration distance from the PAM transposon were mapped and quantified using guideseq = 20 bp LE or RE 3' end, window center = 0, window size = 20. Indel histograms were normalized relative to the total detected indel reads and frequency plotted relative to a 60 bp reference sequence. Figure 14 ).

[0360] PCR reaction 5 (LE proximal to PAM) was plotted based on the sequence of MG64-1 and its distance from PAM. Figure 14 (Top small image) and PCR 4 (RE on the distal side of PAM, Figure 14 (Bottom inset) Both. Analysis of the integration window showed that 95% of the integrations occurring at the spacer PAM site were within a 10 bp window between 58 and 68 nucleotides from PAM. The difference in integration distance between the distal and proximal frequencies reflects integration site repetition—a 3-5 base pair repetition caused by the staggered nuclease activity of transposases during integration.

[0361] Example 19 - Colony PCR Screening for Transposase Activity Transposon activity was determined by colony PCR screening. After transformation with pDonor plasmid, *E. coli* were plated onto LB agar containing ampicillin, chloramphenicol, and tetracycline. Selected CFUs were added to a solution containing PCR reagents and primers flanking the selected insertion sites. The PCR reaction of the integration products was visible on the gel. Figure 15 Sequencing results of the selected colony PCR products confirmed that they represented transposition events, as the selected colony PCR products crossed the junction between LE and PAM at the engineered target site in the lacZ gene. Figure 16 ).

[0362] Example 20 - One-Wizard Engineering Predicted RNA folding for active single RNA sequences was calculated at 37°C using the Andronescu 2007 method. All hairpin loop secondary structures were individually removed from the construct and iteratively compiled into smaller single-guides. Engineered single-guides (esg) 4, 6, 7, 8, and 9 are active for donor transposition. Figure 17 (See figures C and D), where engineered sgRNAs 8 and 9 are weaker single-guided transposons and are transposable using PCR 5 ( Figure 17(See small figure D). Engineered guide 5 can transpose, but engineered sgRNA 10 and PCR 5 transpose more weakly ( Figure 17 (See figures E and F). Esg 17 is a combination of deletions in esg6 and esg7, and esg 18 is a combination of esg 4 and esg 5. Both are capable of strong transposition across PCR 4 and 5. Figure 17 (See figures G and H). However, the combined addition of esg 6 and esg 18 to form esg 19 resulted in weaker transposition in PCR 5, and the addition of esg 7 to esg 19 to form esg 20 resulted in very weak transposition binding sites in PCR 5. Figure 17 (See small figures G and H). In the second method, the tracrRNA of MG64-1 is aligned with the recorded Vk-type tracrRNA, and the uniquely inserted region is mutated to create a single guide. The sgRNA is minimized by truncating the inserted sequence of the MG64-1 sgRNA. Figure 14 Two subsequent missing values ​​were also tested, eg 2 and eg 3 (). Figure 17 (See small figures A and B), but neither esg 2 nor esg 3 causes significant transposition, so the unidirectional guide reduces 57 bases to the maximum extent.

[0363] Example 21 - LE-RE Minimization Sequencing of the target transposon junction, by identifying the outermost sequence from the donor plasmid incorporated into the target reaction, facilitated the identification of terminal inverted repeat sequences. Short repeat sequences contained within the terminals were identified through repeat sequence analysis of a 14 bp segment with 10% variability, and truncation of these minimal ends was designed to preserve repeat sequences while eliminating redundant sequences. Prediction and cloning were performed in multiple iterations, with each interaction tested via in vitro transposition. Initial LE and RE deletions were designed individually and cloned into LEs at 68 bp, 86 bp, and 105 bp, and REs at 178 bp, 196 bp, and 242 bp. The RE at 64-1 also exhibited a significant sequence span without repeat sequences, thus internal deletions of both 50 bp and 81 bp were designed and cloned. Transposition between all individual deletions was performed for PCR 4 and PCR 5. Figure 18 The small figures A and B are robust, and the 81 bp internal deletion was subsequently pursued using the RE combination deletion. The pruned ends of the aforementioned 178 bp, 196 bp, and 212 bp were cloned onto the 81 bp internal deletion, and the transposition was tested. The transposition was active for all constructs of the design. With the 68 bp LE combination, the transposition proved active up to the 68 bp LE region combined with the 96 bp RE region. Figure 18 (Small figures E and F).

[0364] Example 22 - Effect of the protruding end of the transpose To test whether the extra sequence outside the TnsB binding motif is necessary for transposition, oligonucleotides targeting the TGTACA motif for both LE and RE were designed and synthesized with 0, 1, 2, 3, 5, and 10 bp additional base pairs. These synthesized oligonucleotides were used to generate donor PCR fragments with overhangs and their ability to transpose to target sites was tested. Most notably, PCR6 was rarely detected in in vitro reactions. Figure 18 In the small figure G (lanes 1 and 2), however, effective integration at PCR 6 was detected at the 0-3 bp protrusion, reflecting REs that were not detected in the larger side-connected sequence proximal to the PAM orientation.

[0365] Example 23 - CAST NLS Design Eukaryotic genome editing for therapeutic purposes largely relies on the introduction of editing enzymes into the cell nucleus. Small polypeptide fragments of larger proteins signal cellular components for protein transnuclear membrane delivery. Placing these tags is not straightforward, as there is a potential trade-off between delivery function and the function of the fused protein, depending on the location of the NLS tag. To test the functional orientation of NLS for each component of the CAST complex, constructs were designed and synthesized that fused nucleoplasmic NLS to the N-terminus of each component of MG CAST and fused SV40 NLS to the C-terminus. The proteins from these constructs were expressed in cell-free in vitro transcription / translation reactions, and in vitro transposition activity was tested with a set of unlabeled complement components. The maintenance of activity of the NLS-tagged constructs was assessed by PCR at the donor-target junction using PCR 4 (assessing distal RE transposition) and homologous transposition event PCR 5 (LE to proximal transposition).

[0366] Most components produce a single NLS orientation that maintains activity. TnsB is a CAST component that is active against both N-terminal and C-terminal NLS via both PCR4 and PCR5. Figure 19 (Small figures A and B). TniQ is active against N-terminal NLS tags ( Figure 19 Small figures C and D). And the Cas12k component is labeled with NLS at the C-terminus ( Figure 19 The small figures E and F, lanes 5 and 6) showed activity. Further development of Cas12k with both nucleoplasmic protein and SV40 NLS tag was tested, and activity was found ( Figure 19 Small figures I and J, lane 4). TnsC to N-terminal NLS ( Figure 19The small figures E and F, lane 7) showed weak activity, but further exploration of the TnsC label identified new NLS-HA-TnsC and NLS-FLAG-TnsC constructs ( Figure 19 The small figures G and H represent lanes 3 and 7, respectively. The final result is a fully NLS-labeled component kit that is active in vitro for both orientations of NLS-TnsB and TnsB-NLS. Figure 20 Small pictures A and B, lanes 5 and 6).

[0367] Example 24 - Design and Testing of Cas12k and TniQ Protein Fusion Constructs To simplify the expression of protein components and minimize their delivery to cells, fusion constructs between the Cas12k effector and the TniQ protein were designed, synthesized, and tested. Two orientations of TniQ fused to Cas12k were designed and synthesized: C-terminal fusion Cas-TniQ and N-terminal fusion TniQ-Cas. While both constructs were tested for PCR4 (… Figure 21 The small figure (A) shows weak activity, but when expressed in vitro and transposable ability is measured, the PCR5 binding site is robustly formed by the TniQ-Cas fusion protein. Figure 21 (See small figure B). Transposable lengths were determined using variable linker domains containing the original (20 amino acid linkers), 48, 68, 72, and 77. Figure 21 (See figures C, D, E, and F). The NLS tag was then ligated to the N-terminus of TniQ and the C-terminus of Cas12k, and PCR-5 showed it remained active. Figure 21 (Small figures E and F).

[0368] Two additional linkers were used to fuse the effector and the TniQ gene. The P2A, self-stopping translation sequence, was used in the Cas-NLS-P2A-NLS-TniQ construct. Figure 21 The small figures G and H, lane 6) and the ribosome entry sequence (IRES) mRNA-based linker within the MCV are active, and the linker allows the two components to be translated independently in the cell. Figure 23 (Small figures F and G).

[0369] Example 25 - Intracellular Expression Coupled with In vitro Transposition Assay To test the function of the NLS construct in a physiologically relevant setting, constructs containing clones of the active NLS-labeled CAST component were integrated into K562 cells using lentiviral transduction. Briefly, the construct cloned into a lentiviral transfer plasmid was transfected into 293T cells using an encapsulation and packaging plasmid, and the virus containing supernatant was collected from the culture medium after 72 hours of incubation. The virus-containing medium was then incubated with the K562 cell line and 8 µg / mL polybrene for 72 hours. Transfected cells were then selected for bulk integration using 1 µg / mL puromycin over 4 days. At the end of 4 days, the selected cell lines were collected, and differential lysis was performed targeting the nuclear and cytoplasmic fractions. The transposability of subsequent fractions with a set of complementary in vitro expressed components was then tested.

[0370] Both NLS-TnsB and TnsB-NLS were tested by cell grading and in vitro transposition, and transposition of both was detected across the cytoplasm and nuclear gradations. NLS-TniQ also exhibited detectable activity in the cytoplasm. Figure 22 (See figures A and B). When expressed, NLS-HA-TnsC and NLS-FLAG-TnsC are active in both the cytoplasm and nuclear fractions. Figure 22 (See small figure D), however, PCR4 was formed in the nuclear fraction of both TnsC constructs. Figure 22 (small image C).

[0371] When NLS-TnsB or TnsB-NLS is linked to NLS-FLAG-TnsC using IRES, NLS-TnsB-IRES-NLS-FLAG-TnsC is mostly active in the nuclear fraction, while TnsB-NLS-IRES-NLS-FLAG-TnsC is active in both the cytoplasm and nuclear fraction. This indicates that NLS-TnsB has a higher transport capacity to the nucleus. Figure 21 (Small figures E and F).

[0372] Similarly, the Cas12k fusion in cells was fractionated and transposition was tested. Cas-NLS-P2A-NLS-TniQ was transduced into cells, fractionated, and subcellular activity was tested in vitro. Cas-NLS-P2A-NLS-TniQ was able to transpose in the cytoplasm by adding a unidirectional guide to the reaction. Figure 23 (See Figure A). The Cas-NLS-P2A-NLS-TniQ construct in the nuclear fraction is supplemented by adding sgRNA to the holo Cas protein (+sgRNA) or additional TniQ. This instructs both Cas-NLS and NLS-TniQ to form it in the nuclear fraction. Figure 23(See figures B and C). The NLS-TniQ-Cas-NLS fusion protein yielded similar results, but required the use of TniQ (…). Figure 23 The smaller figures D and E) are supplemented further, and Cas-NLS-IRES-NLS-TniQ only requires supplementation from holo Cas-NLS ( Figure 23 (See small figures F and G). Overall, this indicates that all components of CAST are capable of delivery to the nuclear fraction of the cell.

[0373] Example 26 - Transposon End Validation via Gel Translocation To verify the activity of TnsB for predicted transposon terminal sequences, LEs of MG64-1 were amplified using FAM-tagged oligonucleotides. MG64-1 TnsB protein was expressed using a cell-free transcription / translation system and incubated with the FAM-tagged LE product. Binding was observed on a native 5% TBE gel after 30 minutes of incubation. Figure 24 Commonly heated swimming lanes ( Figure 24 Multiple bands of fluorescent products within lane 3 indicate at least two TnsB binding sites.

[0374] The systems disclosed herein can be used for a variety of applications, such as nucleic acid editing (e.g., gene editing) or binding to nucleic acid molecules (e.g., sequence-specific binding). Such systems can be used, for example, to remedy (e.g., remove or replace) genetic mutations that may cause disease in a subject; to inactivate genes to determine their function in cells; as diagnostic tools for detecting pathogenic genetic elements (e.g., by cleaving retroviral RNA or amplified DNA sequences encoding pathogenic mutations); as inactivating enzymes bound to probes to target and detect specific nucleotide sequences (e.g., sequences encoding bacterial antibiotic resistance); to inactivate viruses or prevent them from infecting host cells by targeting viral genomes; to ...

Claims

1. A system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a) A Cas effector complex comprising two types of V-type Cas effectors, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide, wherein the engineered guide polynucleotide hybridizes with the target nucleic acid site; b) A Tn7 transposase complex, said Tn7 transposase complex binding to the Cas effector complex and comprising a TnsB component, a TnsC component, and a TniQ component, and an accessory protein comprising a sequence having at least 70% sequence identity with any one of SEQ ID NO: 228-230 and 235-249; and c) A double-stranded nucleic acid that interacts with the Tn7 transposase complex and includes the cargo nucleotide sequence.

2. The system of claim 1, wherein the Cas effector complex is non-covalently bound to the Tn7 transposase complex.

3. The system according to claim 1, wherein the Cas effector complex is covalently linked to the Tn7 transposase complex.

4. The system of claim 1, wherein the Cas effector complex is fused with the Tn7 transposase complex.

5. The system according to any one of claims 1 to 4, wherein the cargo nucleotide sequence is side-mounted with a left transposase recognition sequence and a right transposase recognition sequence recognized by the Tn7 type transposase complex.

6. The system of claim 5, wherein the left transposase recognition sequence comprises a sequence having at least 80% identity with any one of SEQ ID NO: 9, 11, 36-38, 76 and 78.

7. The system of claim 5, wherein the right-hand transposase recognition sequence comprises a sequence having at least 80% identity with any one of SEQ ID NO: 8, 10, 39-44, 77, 79 and 93.

8. The system according to claims 1 to 7, wherein the target nucleic acid comprises a PAM sequence compatible with the Cas effector complex.

9. The system of claim 8, wherein the PAM sequence comprises SEQ ID NO:

31.

10. The system according to any one of claims 8 to 9, wherein the PAM sequence is located at a distance of about 50 to about 70 base pairs from the target nucleic acid site.

11. The system of claim 10, wherein the PAM sequence is located at the 3' position of the target nucleic acid site.

12. The system of claim 10, wherein the PAM sequence is located at the 5' of the target nucleic acid site.

13. The system according to any one of claims 1 to 12, wherein the type 2 V-type Cas effector is a Cas12k effector.

14. The system according to any one of claims 1 to 12, wherein the type 2 V-type Cas effector comprises a polypeptide containing a sequence having at least 80% identity with any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85 and 220.

15. The system according to any one of claims 1 to 12, wherein the type 2 V-type Cas effector comprises a polypeptide containing a sequence having at least 90% identity with any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85 and 220.

16. The system according to any one of claims 1 to 12, wherein the type 2 V-type Cas effector comprises a polypeptide comprising a sequence of any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85 and 220.

17. The system according to any one of claims 1 to 16, wherein the TnsB component comprises a polypeptide having a sequence having at least 80% identity with any one of SEQ ID NO: 2, 13, 17 and 65.

18. The system according to any one of claims 1 to 16, wherein the TnsB component comprises a polypeptide having a sequence having at least 90% identity with any one of SEQ ID NO: 2, 13, 17 and 65.

19. The system according to any one of claims 1 to 16, wherein the TnsB component comprises a polypeptide having the sequence of any one of SEQ ID NO: 2, 13, 17 and 65.

20. The system according to any one of claims 1 to 19, wherein the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently comprising a sequence having at least 80% identity with any one of SEQ ID NO: 3-4, 14-15, 18-19, 66-67 and 109-111.

21. The system according to any one of claims 1 to 19, wherein the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently comprising a sequence having at least 90% identity with any one of SEQ ID NO: 3-4, 14-15, 18-19, 66-67 and 109-111.

22. The system according to any one of claims 1 to 19, wherein the Tn7 transposase complex comprises at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently comprising the sequence of any one of SEQ ID NO: 3-4, 14-15, 18-19, 66-67 and 109-111.

23. The system according to any one of claims 1 to 22, wherein the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least 80% identity with any one of SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122 and 222.

24. The system according to any one of claims 1 to 22, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140 and 754-944.

25. The system according to any one of claims 1 to 23, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 187-189.

26. The system according to any one of claims 1 to 23, wherein the small prokaryotic ribosomal protein subunit S15 is encoded by a sequence having at least 80% sequence identity with any one of SEQ ID NO: 181-183.

27. The system according to any one of claims 1 to 26, wherein the type V Cas effector and the Tn7 transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

28. The system according to any one of claims 1 to 27, wherein the accessory protein is ClpX, said ClpX comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 235-249.

29. A system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a) A Cas effector complex comprising two types of V-type Cas effectors, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide, wherein the engineered guide polynucleotide hybridizes with the target nucleic acid site; b) A Tn7 transposase complex, said Tn7 transposase complex being bound to said Cas effector complex and comprising a functional domain (FD)-TniQ fusion and an accessory protein; and c) A double-stranded nucleic acid that interacts with the Tn7 transposase complex and includes the cargo nucleotide sequence.

30. The system of claim 29, wherein the Cas effector complex is non-covalently bound to the Tn7 transposase complex.

31. The system of claim 29, wherein the Cas effector complex is covalently linked to the Tn7 transposase complex.

32. The system of claim 29, wherein the Cas effector complex is fused with the Tn7 transposase complex.

33. The system of claim 29, wherein the functional structural domain (FD) comprises a sequence having at least 80% identity with any one of SEQ ID NO: 257-307 and 1138-1242.

34. The system according to any one of claims 29 to 33, wherein the cargo nucleotide sequence is flanked by a left transposase recognition sequence and a right transposase recognition sequence recognized by the Tn7 type transposase complex.

35. The system of claim 34, wherein the left transposase recognition sequence comprises a sequence having at least 80% identity with any one of SEQ ID NO: 9, 11, 36-38, 76 and 78.

36. The system according to any one of claims 34, wherein the right-hand transposase recognition sequence comprises a sequence having at least 80% identity with any one of SEQ ID NO: 8, 10, 39-44, 77, 79 and 93.

37. The system of claims 29 to 36, wherein the target nucleic acid comprises a PAM sequence compatible with the Cas effector complex.

38. The system of claim 37, wherein the PAM sequence comprises SEQ ID NO:

31.

39. The system according to any one of claims 37 to 38, wherein the PAM sequence is located at a distance of about 50 to about 70 base pairs from the target nucleic acid site.

40. The system of claim 39, wherein the PAM sequence is located at the 3' position of the target nucleic acid site.

41. The system of claim 40, wherein the PAM sequence is located at the 5' of the target nucleic acid site.

42. The system according to any one of claims 29 to 41, wherein the type 2 V-type Cas effector is a Cas12k effector.

43. The system according to any one of claims 29 to 42, wherein the type V Cas effector comprises a polypeptide having a sequence having at least 90% identity with any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85 and 220.

44. The system according to any one of claims 29 to 43, wherein the type 2 V-type Cas effector comprises a polypeptide comprising a sequence of any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85 and 220.

45. The system according to any one of claims 29 to 44, wherein the engineered guide polynucleotide comprises a sequence having at least 80% identity with any one of SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122 and 222, consisting of at least about 46-80 consecutive nucleotides.

46. ​​The system according to any one of claims 29 to 45, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140 and 754-944.

47. The system according to any one of claims 29 to 46, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 187-189.

48. The system according to any one of claims 29 to 46, wherein the small prokaryotic ribosomal protein subunit S15 is encoded by a sequence having at least 80% sequence identity with any one of SEQ ID NO: 181-183.

49. The system according to any one of claims 29 to 48, wherein the type V Cas effector and the Tn7 transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

50. The system according to any one of claims 29 to 49, wherein the accessory protein comprises a sequence having at least 70% sequence identity with any one of SEQ ID NO: 228-230 and 235-249.

51. The system of claim 50, wherein the accessory protein is ClpX, wherein ClpX comprises a sequence having at least 80% sequence identity with any of SEQ ID NO: 235-249.

52. A system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a) A Cas effector complex comprising two types of V Cas effectors and an engineered guide polynucleotide, the engineered guide polynucleotide hybridizing with the target nucleic acid site, wherein the Cas effector complex comprises a polypeptide containing a sequence having at least 80% sequence identity with any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85 and 220; b) A Tn7 transposase complex, said Tn7 transposase complex being bound to the Cas effector complex and comprising a TnsB component, a TnsC component, and a TniQ component, and an accessory protein, wherein the TnsB component, the TnsC component, or the TniQ component comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 2-4, 13-15, 17-19, 65-67, and 109-111, and the accessory protein comprises a sequence having at least 70% sequence identity with any one of SEQ ID NO: 228-230 and 235-249; and c) A double-stranded nucleic acid that interacts with the Tn7 transposase complex and includes the cargo nucleotide sequence.

53. The system of claim 29, wherein the Cas effector complex is non-covalently bound to the Tn7 transposase complex.

54. The system of claim 29, wherein the Cas effector complex is covalently linked to the Tn7 transposase complex.

55. The system of claim 29, wherein the Cas effector complex is fused with the Tn7 transposase complex.

56. The system according to any one of claims 29 to 55, wherein the cargo nucleotide sequence is side-mounted with a left transposase recognition sequence and a right transposase recognition sequence recognized by the Tn7 type transposase complex.

57. The system of claim 56, wherein the left transposase recognition sequence comprises a sequence having at least 80% identity with any one of SEQ ID NO: 9, 11, 36-38, 76 and 78.

58. The system according to any one of claims 56, wherein the right-hand transposase recognition sequence comprises a sequence having at least 80% identity with any one of SEQ ID NO: 8, 10, 39-44, 77, 79 and 93.

59. The system according to claims 29 to 58, wherein the target nucleic acid comprises a PAM sequence compatible with the Cas effector complex.

60. The system of claim 59, wherein the PAM sequence comprises SEQ ID NO:

31.

61. The system according to any one of claims 59 to 60, wherein the PAM sequence is located at a distance of about 50 to about 70 base pairs from the target nucleic acid site.

62. The system of claim 61, wherein the PAM sequence is located at the 3' position of the target nucleic acid site.

63. The system of claim 61, wherein the PAM sequence is located at the 5' of the target nucleic acid site.

64. The system according to any one of claims 29 to 63, wherein the type 2 V-type Cas effector is a Cas12k effector.

65. The system according to any one of claims 29 to 63, wherein the type V Cas effector comprises a polypeptide having a sequence having at least 90% identity with any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85 and 220.

66. The system according to any one of claims 29 to 63, wherein the type 2 V-type Cas effector comprises a polypeptide comprising a sequence of any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85 and 220.

67. The system according to any one of claims 29 to 63, wherein the TnsB component, the TnsC component, or the TniQ component comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 2-4, 13-15, 17-19, 65-67, and 109-111.

68. The system according to any one of claims 29 to 63, wherein the TnsB component, the TnsC component, or the TniQ component comprises a sequence of any one of SEQ ID NO: 2-4, 13-15, 17-19, 65-67, and 109-111.

69. The system according to any one of claims 29 to 68, wherein the engineered guide polynucleotide comprises a sequence containing at least about 46-80 consecutive nucleotides having at least 80% identity with any one of SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122 and 222.

70. The system according to any one of claims 29 to 68, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 106, 107, 108, 5, 45-63, 68-75, 96-103, 123-140 and 754-944.

71. The system according to any one of claims 29 to 70, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 187-189.

72. The system according to any one of claims 29 to 70, wherein the small prokaryotic ribosomal protein subunit S15 is encoded by a sequence having at least 80% sequence identity with any one of SEQ ID NO: 181-183.

73. The system according to any one of claims 29 to 72, wherein the type V Cas effector and the Tn7 transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

74. The system according to any one of claims 29 to 73, wherein the accessory protein is ClpX, said ClpX comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 235-249.

75. A system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a) A Cas effector complex, which hybridizes to the target nucleic acid site and comprises: i) Two types of V-type Cas effectors, wherein the two types of V-type Cas effectors include sequences having at least 80% sequence identity with any one of SEQ ID NO: 1, 81, 82, 83 and 85; and ii) an engineered guide polynucleotide having at least 80% identity with any one of SEQ ID NO: 5, 6, 45-63, 68-75, 96-103, 123-140 and 754-944; b) A Tn7 transposase complex, said Tn7 transposase complex being bound to the Cas effector complex and comprising a TnsB component, a TnsC component, and a TniQ component, and an accessory protein, wherein the TnsB component, the TnsC component, or the TniQ component comprises a sequence having at least 80% identity with any of SEQ ID NO: 2-4, and the accessory protein comprises a sequence having at least 70% sequence identity with any of SEQ ID NO: 228-230 and 235-249; and c) A double-stranded nucleic acid that interacts with the Tn7 transposase complex and comprises, in a 5' to 3' order: i) A left transposase recognition sequence, wherein the left transposase recognition sequence comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 9, 11, 36, 37 and 38; ii) the nucleotide sequence of the cargo; and ii) Right-sided transposase recognition sequence, said right-sided transposase recognition sequence comprising a sequence having at least 80% identity with any one of SEQ ID NO: 8, 39-44 and 93.

76. A system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a) A Cas effector complex, which hybridizes to the target nucleic acid site and comprises: i) Two types of V-type Cas effectors, wherein the two types of V-type Cas effectors include sequences having at least 80% sequence identity with SEQ ID NO: 12; and iii) an engineered guide polynucleotide having at least 80% identity with any one of SEQ ID NO: 32, 102, 104 and 107; b) A Tn7 transposase complex, said Tn7 transposase complex being bound to the Cas effector complex and comprising a TnsB component, a TnsC component, and a TniQ component, and an accessory protein, wherein the TnsB component, the TnsC component, or the TniQ component comprises a sequence having at least 80% identity with any of SEQ ID NO: 13-15, and the accessory protein comprises a sequence having at least 70% sequence identity with any of SEQ ID NO: 228-230 and 235-249; and c) A double-stranded nucleic acid that interacts with the Tn7 transposase complex and comprises, in a 5' to 3' order: i) A left transposase recognition sequence, wherein the left transposase recognition sequence comprises a sequence having at least 80% sequence identity with SEQ ID NO: 76; ii) the nucleotide sequence of the cargo; and iii) Right-sided transposase recognition sequence, wherein the right-sided transposase recognition sequence comprises a sequence having at least 80% identity with SEQ ID NO:

77.

77. A system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a) A Cas effector complex, which hybridizes to the target nucleic acid site and comprises: i) Two types of V-type Cas effectors, wherein the two types of V-type Cas effectors include sequences having at least 80% sequence identity with SEQ ID NO: 16; and ii) an engineered guide polynucleotide having at least 80% identity with any one of SEQ ID NO: 33, 103, 105 and 108; b) A Tn7 transposase complex, said Tn7 transposase complex being bound to the Cas effector complex and comprising a TnsB component, a TnsC component, and a TniQ component, and an accessory protein, wherein the TnsB component, the TnsC component, or the TniQ component comprises a sequence having at least 80% identity with any of SEQ ID NO: 17-19, and the accessory protein comprises a sequence having at least 70% sequence identity with any of SEQ ID NO: 228-230 and 235-249; and c) A double-stranded nucleic acid that interacts with the Tn7 transposase complex and comprises, in a 5' to 3' order: i) A left transposase recognition sequence, wherein the left transposase recognition sequence comprises a sequence having at least 80% sequence identity with SEQ ID NO: 78; ii) the nucleotide sequence of the cargo; and iii) Right-sided transposase recognition sequence, wherein the right-sided transposase recognition sequence includes a sequence having at least 80% identity with SEQ ID NO:

79.

78. The system according to any one of claims 75 to 77, further comprising a PAM sequence compatible with the Cas effector complex.

79. The system of claim 78, wherein the PAM sequence comprises SEQ ID NO:

31.

80. The system according to any one of claims 78 to 79, wherein the PAM sequence is located at a distance of about 50 to about 70 base pairs from the target nucleic acid site.

81. The system of claim 80, wherein the PAM sequence is located at the 3' position of the target nucleic acid site.

82. The system of claim 80, wherein the PAM sequence is located at the 5' of the target nucleic acid site.

83. The system according to any one of claims 75 to 82, wherein the Cas effector complex further comprises the small prokaryotic ribosomal protein subunit S15.

84. The system of claim 83, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity with any of SEQ ID NO: 187-189.

85. The system according to any one of claims 75 to 84, wherein the accessory protein is ClpX, said ClpX comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 235-249.

86. An engineered nuclease system comprising: a) an endonuclease comprising a RuvC domain, said endonuclease being derived from uncultured microorganisms and being a type 2 VK Cas effector, said type 2 VK Cas effector sharing at least 80% identity with any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220; and b) An engineered guide RNA, wherein the engineered guide RNA forms a complex with the endonuclease and includes a spacer sequence that hybridizes with a target nucleic acid sequence, wherein the engineered guide polynucleotide includes a sequence that is at least 80% identical to any of SEQ ID NO: 754-944.

87. A method for transposing a cargo nucleotide sequence into a target nucleic acid site, the method comprising introducing the system according to any one of claims 1 to 86 into a cell.

88. A cell comprising the system according to any one of claims 1 to 86.

89. The cell of claim 88, wherein the cell is a eukaryotic cell.

90. The cell of claim 88, wherein the cell is a mammalian cell.

91. The cell of claim 88, wherein the cell is an immortalized cell.

92. The cell according to claim 88, wherein the cell is an insect cell.

93. The cell according to claim 88, wherein the cell is a yeast cell.

94. The cell of claim 88, wherein the cell is a plant cell.

95. The cell of claim 88, wherein the cell is a fungal cell.

96. The cell of claim 88, wherein the cell is a prokaryotic cell.

97. The cell according to claim 88, wherein the cell is A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cells, HT1080, HepG2, Huh7, K562, primary cells or derivatives thereof.

98. The cell of claim 88, wherein the cell is an engineered cell.

99. The cell of claim 88, wherein the cell is a stable cell.

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