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 is configured to interact with a recombinase complex; an effector complex comprising an effector and at least one engineered guide polynucleotide configured to hybridize to the target nucleic acid; and the recombinase complex, wherein the recombinase complex is configured to recruit the cargo nucleotide to the target nucleic acid site.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 313,156, filed on February 23, 2022, and U.S. Provisional Patent Application No. 63 / 478,689, filed on January 5, 2023, each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] The contents of the electronic sequence listing (MTG-008WO_SL.xml; size: 223,433 bytes; and creation date: February 23, 2023) are incorporated herein by reference in their entirety. Background Art

[0005] Cas enzymes and their associated clustered regularly interspaced short palindromic repeats (CRISPR) guide RNAs appear to be universal components of the prokaryotic immune system (~45% of bacteria, ~84% of archaea), used to protect such microorganisms from non-self nucleic acids, such as infectious viruses and plasmids, through 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 capabilities of the CRISPR / Cas complex were not recognized until recently, leading to the use of recombinant CRISPR / Cas systems in various DNA manipulation and gene editing applications. Summary of the invention

[0006] In some aspects, the present 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, the first double-stranded nucleic acid comprising a cargo nucleotide sequence configured to interact with a Tn7-type transposase complex; a Cas effector complex, the Cas effector complex comprising a 2-type V-type Cas effector and an engineered guide polynucleotide, the engineered guide polynucleotide being configured to hybridize with the target nucleotide sequence; and a Tn7-type transposase complex, the Tn7-type transposase complex being 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 transposase recognition sequence and a right transposase recognition sequence. In some embodiments, the system further comprises a second double-stranded nucleic acid comprising 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 3' of the target nucleic acid site. In some embodiments, the PAM sequence is located at 5' of the target nucleic acid site. In some embodiments, the engineered guide polynucleotide is configured to bind to the Class 2 Type V Cas effector. In some embodiments, the Class 2 Type V Cas effector comprises a polypeptide comprising a sequence having at least 80% identity to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220 or a variant thereof. In some embodiments, the TnsB subunit comprises a polypeptide having a sequence having at least 80% identity to SEQ ID NO: 2, 13, 17, and 65 or a variant thereof. In some embodiments, the Tn7-type transposase complex comprises at least one, at least two, or three polypeptides comprising a sequence having at least 80% identity to any one of SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111 or a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least 80% identity to any one of SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222, or variants thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to non-degenerate nucleotides of any one of SEQ ID NOs: 106, 107, 108, 5, 45-63, 68-75, 96-103, or 123-140, or variants thereof. In some embodiments, the left recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78, or variants thereof.In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 8, 10, 39-44, 77, 79 and 93 or a variant thereof. In some embodiments, the class 2 type V Cas effector and the Tn7 type transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0007] In some aspects, the disclosure provides a method for transposing a cargo nucleotide sequence into a target nucleic acid site comprising a target nucleotide sequence, the method comprising expressing the system of any aspect or embodiment described herein in a cell or introducing the system of any aspect or embodiment described herein into a cell.

[0008] In some aspects, the present disclosure provides a method for transposing a cargo nucleotide sequence into a target nucleic acid site, the method comprising contacting a first double-stranded nucleic acid comprising the cargo nucleotide sequence with: a Cas effector complex comprising 2 types of V-type Cas effectors and at least one engineered guide polynucleotide, the at least one engineered guide polynucleotide being configured to hybridize with the target nucleotide sequence; a Tn7-type transposase complex, the Tn7-type transposase complex being configured to bind to the Cas effector complex, wherein the Tn7-type transposase complex comprises a TnsB subunit; and a second double-stranded nucleic acid comprising the target nucleic acid site. In some embodiments, the cargo nucleotide sequence is flanked by a left transposase recognition sequence and a right transposase recognition sequence. 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 3' of the target nucleic acid site. In some embodiments, the engineered guide polynucleotide is configured to bind to the 2 types of V-type Cas effectors. In some embodiments, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least 80% identity to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85 and 220 or a variant thereof. In some embodiments, the TnsB subunit comprises a polypeptide having a sequence having at least 80% identity to SEQ ID NO: 2, 13, 17 and 65 or a variant thereof. In some embodiments, the Tn7-type transposase complex comprises at least one or at least two polypeptides comprising a sequence having at least 80% identity to any one of SEQ ID NO: 3-4, 14-15, 18-19, 66-67 and 109-111 or a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least 80% identity to any one of SEQ ID NO: 5-6, 32-33, 94-95, 104-105, 119-122 and 222 or a variant thereof. In some embodiments, the left recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 9, 11, 36-38, 76 and 78 or a variant thereof. In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 8, 10, 39-44, 77, 79 and 93 or a variant thereof. In some embodiments, the class 2 type V Cas effector and the Tn7 type transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0009] In some aspects, the present 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, the first double-stranded nucleic acid comprising a cargo nucleotide sequence configured to interact with a Tn7-type transposase complex; a Cas effector complex, the Cas effector complex comprising a 2-type V-type Cas effector and an engineered guide polynucleotide, the engineered guide polynucleotide being configured to hybridize with the target nucleotide sequence; and a Tn7-type transposase complex, the Tn7-type transposase complex being configured to bind to the Cas effector complex, wherein the Tn7-type transposase complex comprises TnsB, TnsC, and TniQ components, wherein: (a) the 2-type V-type Cas effector comprises a polypeptide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220, or a variant thereof; (b) the Tn7-type transposase complex comprises a polypeptide having a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220, or a variant thereof; NO: 2-4, 13-15, 17-19, 65-67 and 109-111 or variants thereof having a sequence of at least 80% sequence identity TnsB, TnsC and TniQ components. In some embodiments, the transposase complex is non-covalently bound to the Cas effector complex. In some embodiments, the transposase complex is covalently linked to the Cas effector complex. In some embodiments, the transposase complex is fused to the Cas effector complex in a single polypeptide. In some embodiments, the Class 2 V-type Cas effector comprises a polypeptide having a sequence having at least 80% sequence identity to any one of SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85 and 220 or variants thereof. In some embodiments, the Tn7-type transposase complex includes TnsB, TnsC and TniQ components having a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 2-4, 13-15, 17-19, 65-67 and 109-111 or a variant thereof. In some embodiments, the 2 types of V-type Cas effectors are Cas12k effectors. In some embodiments, the cargo nucleotide sequence is flanked by a left transposase recognition sequence and a right transposase recognition sequence. In some embodiments, the system further includes a second double-stranded nucleic acid comprising the target nucleic acid site. In some embodiments, the system 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 5' or 3' of the target nucleic acid site. In some embodiments, the PAM sequence includes SEQ ID NO: 31. In some embodiments, the engineered guide polynucleotide is configured to bind to the 2 types of V-type Cas effectors.In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least 80% identity to any one of SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222, or variants thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to non-degenerate nucleotides of any one of SEQ ID NOs: 106, 107, 108, 5, 45-63, 68-75, 96-103, or 123-140, or variants thereof. In some embodiments, the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 9, 11, 36-38, 76, and 78, or variants thereof. In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some embodiments, the 2-type V-type Cas effector and the Tn7-type transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases. In some embodiments: (a) the 2-type V-type Cas effector comprises a sequence having at least 80% sequence identity to any one of SEQ ID NO: 1, 81, 82, 83 or 85 or a variant thereof; (b) the left recombinase sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NO: 9, 11, 36, 37 or 38 or a variant thereof; (c) the right recombinase sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NO: 8, 39, 40, 41, 42, 43, 44 or 93 or a variant thereof; (d) the engineered guide polynucleotide: (i) comprises a sequence having at least 80% sequence identity to at least about 46-80 nucleotides of SEQ ID NO: 6 or a variant thereof; or (ii) comprises a sequence having at least 80% sequence identity to SEQ ID NO: 9, 11, 36, 37 or 38 or a variant thereof; NO:5, 45-63, 68-75, 96-103 or 123-140 or its variants have a sequence with at least 80% identity to the non-degenerate nucleotides of any one of NO:5, 45-63, 68-75, 96-103 or 123-140 or its variants; (e) the TnsB, TnsC and TniQ components include a polypeptide having a sequence with at least 80% identity to SEQ ID NO:2-4 or its variants; or (f) the PAM sequence includes SEQ ID NO:31.In some embodiments: (a) the Class 2 V-type Cas effector comprises a sequence having at least 80% sequence identity with SEQ ID NO: 12 or a variant thereof; (b) the left recombinase sequence comprises a sequence having at least 80% sequence identity with SEQ ID NO: 76 or a variant thereof; (c) the right recombinase sequence comprises a sequence having at least 80% identity with SEQ ID NO: 77 or a variant thereof; (d) the engineered guide polynucleotide: (i) comprises a sequence having at least 80% sequence identity with at least about 46-80 nucleotides of SEQ ID NO: 32 or 104 or a variant thereof; or (ii) comprises a sequence having at least 80% identity with non-degenerate nucleotides of any one of SEQ ID NO: 107 or 102 or a variant thereof; (e) the TnsB, TnsC and TniQ components comprise polypeptides having sequences having at least 80% identity with SEQ ID NO: 13-15 or a variant thereof. In some embodiments: (a) the Class 2 V-type Cas effector comprises a sequence having at least 80% sequence identity with SEQ ID NO: 16 or a variant thereof; (b) the left recombinase sequence comprises a sequence having at least 80% sequence identity with SEQ ID NO: 78 or a variant thereof; (c) the right recombinase sequence comprises a sequence having at least 80% identity with SEQ ID NO: 79 or a variant thereof; (d) the engineered guide polynucleotide: (i) comprises a sequence having at least 80% sequence identity with at least about 46-80 nucleotides of SEQ ID NO: 33 or 105 or a variant thereof; or (ii) comprises a sequence having at least 80% identity with non-degenerate nucleotides of any one of SEQ ID NO: 108 or 103 or a variant thereof; (e) the TnsB, TnsC and TniQ components comprise polypeptides having sequences having at least 80% identity with SEQ ID NO: 17-19 or a variant thereof.

[0010] In some aspects, the present disclosure provides an engineered nuclease system comprising: an endonuclease comprising a RuvC domain, wherein the endonuclease is derived from an uncultured microorganism, and wherein the endonuclease is a class 2 VK-type Cas effector having at least 80% identity to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220, or a variant thereof; and an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222, or a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% identity to a non-degenerate nucleotide of any one of SEQ ID NOs: 106, 107, 108, 5, 45-63, 68-75, 96-103, or 123-140, or a variant thereof. In some embodiments, the system further comprises a PAM sequence adjacent to the target nucleic acid site that is compatible with the Cas effector complex. In some embodiments, the PAM sequence is located 5' of the target nucleic acid site. In some embodiments, the PAM sequence comprises SEQ ID NO: 31. In some embodiments: (a) the class 2 VK-type Cas effector comprises a sequence having at least 80% sequence identity to any one of SEQ ID NO: 1, 81, 82, 83 or 85 or a variant thereof; (b) the left recombinase sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NO: 9, 11, 36, 37 or 38 or a variant thereof; (c) the right recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NO: 8, 39, 40, 41, 42, 43, 44 or 93 or a variant thereof; (d) the engineered guide polynucleotide: (i) comprises a sequence having at least 80% sequence identity to at least about 46-80 nucleotides of SEQ ID NO: 6 or a variant thereof; or (ii) comprises a sequence having at least 80% sequence identity to SEQ ID NO: 7 or a variant thereof; NO:5, 45-63, 68-75, 96-103 or 123-140 or its variants have a sequence with at least 80% identity to the non-degenerate nucleotides of any one of NO:5, 45-63, 68-75, 96-103 or 123-140 or its variants; (e) the TnsB, TnsC and TniQ components include a polypeptide having a sequence with at least 80% identity to, SEQ ID NO:2-4 or its variants; or (f) the PAM sequence includes SEQ ID NO:31.

[0011] In some aspects, the present 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, the Cas effector complex comprising a class 2 V-type Cas effector, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide configured to hybridize with a target nucleic acid site; a Tn7 transposase complex, the Tn7 transposase complex being configured to bind to the Cas effector complex and comprising TnsB, TnsC, and TniQ components; and a double-stranded nucleic acid configured to interact with the Tn7 transposase complex and comprising a cargo nucleotide sequence. 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.

[0012] In some embodiments, the cargo nucleotide sequence is flanked by a left transposase recognition sequence and a right transposase recognition sequence recognized by a Tn7-type transposase complex. In some embodiments, the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93.

[0013] In some embodiments, the target nucleic acid comprises a PAM sequence that is compatible with the Cas effector complex. In some embodiments, the PAM sequence comprises SEQ ID NO: 31. In some embodiments, the PAM sequence is located about 50 to about 70 base pairs from the target nucleic acid site. In some embodiments, the PAM sequence is located 3' of the target nucleic acid site. In some embodiments, the PAM sequence is located 5' of the target nucleic acid site.

[0014] In some embodiments, the Class 2 type V Cas effector is a Cas12k effector. In some embodiments, the Class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the Class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the Class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the TnsB component comprises a polypeptide having a sequence having at least 80% identity to any one of SEQ ID NOs: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide having a sequence at least 90% identical to any one of SEQ ID NOs: 2, 13, 17, and 65. In some embodiments, the TnsB component comprises a polypeptide having a sequence of any one of SEQ ID NOs: 2, 13, 17, and 65. In some embodiments, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence at least 80% identical to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence at least 90% identical to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each independently include the sequence of any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111.

[0015] In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least 80% identity to any one of SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 106, 107, 108, 5, 45-63, 68-75, 96-103, and 123-140.

[0016] In some embodiments, the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 187-189. In some embodiments, the small prokaryotic ribosomal protein subunit S15 is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 181-183.

[0017] In some embodiments, the class 2 type V Cas effector and Tn7-type transposase complex is encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0018] In some aspects, the present 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 a class 2 type V Cas effector and an engineered guide polynucleotide configured to hybridize to the target nucleic acid site, wherein the Cas effector complex comprises a polypeptide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220; a Tn7-type transposase complex configured to bind to the Cas effector complex and comprising TnsB, TnsC, and TniQ components; the TnsB, TnsC, or TniQ components comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2-4, 13-15, 17-19, 65-67, and 109-111; and a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex and comprising the cargo nucleotide sequence.

[0019] In some embodiments, the Cas effector complex is non-covalently bound to the Tn7-type transposase complex. In some embodiments, the Cas effector complex is covalently linked to the Tn7-type transposase complex. In some embodiments, the Cas effector complex is fused to the Tn7-type transposase complex.

[0020] In some embodiments, the cargo nucleotide sequence is flanked by a left transposase recognition sequence and a right transposase recognition sequence recognized by a Tn7-type transposase complex. In some embodiments, the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93.

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

[0022] In some embodiments, the class 2 type V Cas effector is a Cas12k effector. In some embodiments, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the class 2 type V Cas effector comprises a polypeptide comprising a sequence of any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220.

[0023] In some embodiments, the TnsB, TnsC or TniQ component comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2-4, 13-15, 17-19, 65-67 and 109-111. In some embodiments, the TnsB, TnsC or TniQ component comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2-4, 13-15, 17-19, 65-67 and 109-111.

[0024] In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least 80% identity to any one of SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 106, 107, 108, 5, 45-63, 68-75, 96-103, and 123-140.

[0025] In some embodiments, the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 187-189. In some embodiments, the small prokaryotic ribosomal protein subunit S15 is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 181-183.

[0026] In some embodiments, the class 2 type V Cas effector and Tn7-type transposase complex is encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0027] In some aspects, the present 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, the Cas effector complex being configured to hybridize to the target nucleic acid site and comprising: 2 class V-type Cas effectors, the 2 class V-type Cas effectors comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 81, 82, 83, and 85, and an engineered guide polynucleotide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 5, 6, 45-63, 68-75, 96-103, and 123-140; a Tn7-type transposase complex, the Tn7-type transposase complex being configured to bind to the Cas effector complex and comprising TnsB, TnsC, and TniQ components, the TnsB, TnsC, or TniQ components comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: NO:2-4 having at least 80% identity; and a double-stranded nucleic acid configured to interact with a Tn7-type transposase complex and comprising, in 5' to 3' order: a left recombinase sequence comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO:9, 11, 36, 37 and 38; a cargo nucleotide sequence; and a right recombinase sequence comprising a sequence having at least 80% identity with any one of SEQ ID NO:8, 39-44 and 93.

[0028] In some aspects, the present 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, the Cas effector complex being configured to hybridize to the target nucleic acid site and comprising: a 2-type V-type Cas effector, the 2-type V-type Cas effector comprising a sequence having at least 80% sequence identity to SEQ ID NO: 12; and an engineered guide polynucleotide, the engineered guide polynucleotide comprising at least 80% identity to any one of SEQ ID NO: 32, 102, 104, and 107; a Tn7-type transposase complex, the Tn7-type transposase complex being configured to bind to the Cas effector complex and comprising TnsB, TnsC, and TniQ components, the TnsB, TnsC, or TniQ components comprising a sequence having at least 80% sequence identity to SEQ ID NO: NO:13-15 having at least 80% identity; and a double-stranded nucleic acid configured to interact with a Tn7-type transposase complex and comprising, in 5' to 3' order: a left-side recombinase sequence comprising a sequence having at least 80% sequence identity with SEQ ID NO:76; a cargo nucleotide sequence; and a right-side recombinase sequence comprising a sequence having at least 80% identity with SEQ ID NO:77.

[0029] In some aspects, the present 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, the Cas effector complex being configured to hybridize to the target nucleic acid site and comprising: 2 class V-type Cas effectors, the 2 class V-type Cas effectors comprising a sequence having at least 80% sequence identity to SEQ ID NO: 16; and an engineered guide polynucleotide comprising at least 80% identity to any one of SEQ ID NO: 33, 103, 105, and 108; a Tn7-type transposase complex, the Tn7-type transposase complex being configured to bind to the Cas effector complex and comprising TnsB, TnsC, and TniQ components, the TnsB, TnsC, or TniQ components comprising a sequence having at least 80% sequence identity to SEQ ID NO: 16; and an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NO: 33, 103, 105, and 108; and a Tn7-type transposase complex. NO:17-19 having at least 80% identity; and a double-stranded nucleic acid configured to interact with a Tn7-type transposase complex and comprising, in 5' to 3' order: a left-side recombinase sequence comprising a sequence having at least 80% sequence identity with SEQ ID NO:78; a cargo nucleotide sequence; and a right-side recombinase sequence comprising a sequence having at least 80% identity with SEQ ID NO:79.

[0030] In some aspects, the present 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, the Cas effector complex being configured to hybridize to the target nucleic acid site and comprising: 2 class V-type Cas effectors, the 2 class V-type Cas effectors comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 81, 82, 83, and 85, and an engineered guide polynucleotide comprising at least 80% identity to any one of SEQ ID NOs: 5, 6, 45-63, 68-75, 96-103, or 123-140; a Tn7-type transposase complex, the Tn7-type transposase complex being configured to bind to the Cas effector complex and comprising TnsB, TnsC, and TniQ components, the TnsB, TnsC, or TniQ components comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: NO:2-4 having at least 80% identity; and a double-stranded nucleic acid configured to interact with a Tn7-type transposase complex and comprising, in 5' to 3' order: a left recombinase sequence comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO:9, 11, 36, 37 and 38; a cargo nucleotide sequence; and a right recombinase sequence comprising a sequence having at least 80% identity with SEQ ID NO:8, 39-44 and 93.

[0031] In some embodiments, the system further comprises a PAM sequence compatible with the Cas effector complex. In some embodiments, the PAM sequence comprises SEQ ID NO: 31.

[0032] In some embodiments, the PAM sequence is located about 50 to about 70 base pairs from the target nucleic acid site. In some embodiments, the PAM sequence is located 3' of the target nucleic acid site. In some embodiments, the PAM sequence is located 5' of the target nucleic acid site.

[0033] 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 to any one of SEQ ID NOs: 187-189. In some embodiments, the small prokaryotic ribosomal protein subunit S15 is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 181-183.

[0034] In some aspects, the present 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, the Cas effector complex comprising a class 2 type V Cas effector, 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-type transposase complex, the Tn7-type transposase complex being operably linked to the Cas effector complex and comprising TnsB, TnsC, and TniQ components; and a double-stranded nucleic acid comprising, in 5' to 3' order: a left recombinase recognition sequence; a cargo nucleotide sequence; and a right recombinase recognition sequence, wherein the left recombinase recognition sequence and the right recombinase recognition sequence are recognized by the Tn7-type transposase complex.

[0035] In some embodiments, the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93.

[0036] In some embodiments, the target nucleic acid comprises a PAM sequence that is compatible with the Cas effector complex. In some embodiments, the PAM sequence comprises SEQ ID NO: 31. In some embodiments, the PAM sequence is located about 50 to about 70 base pairs from the target nucleic acid site. In some embodiments, the PAM sequence is located 3' of the target nucleic acid site. In some embodiments, the PAM sequence is located 5' of the target nucleic acid site.

[0037] In some embodiments, the class 2 type V Cas effector is a Cas12k effector. In some embodiments, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some embodiments, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220.

[0038] In some embodiments, the TnsB subunit comprises a polypeptide having a sequence at least 80% identical to any one of SEQ ID NOs: 2, 13, 17, and 65. In some embodiments, the TnsB subunit comprises a polypeptide having a sequence at least 90% identical to any one of SEQ ID NOs: 2, 13, 17, and 65. In some embodiments, the TnsB subunit comprises a polypeptide having a sequence of any one of SEQ ID NOs: 2, 13, 17, and 65. In some embodiments, the TnsB subunit comprises a polypeptide having a sequence of any one of SEQ ID NOs: 2, 13, 17, and 65. In some embodiments, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence at least 80% identical to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each independently comprise a sequence having at least 90% identity to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each independently comprise a sequence of any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111.

[0039] In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least 80% identity to any one of SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 106, 107, 108, 5, 45-63, 68-75, 96-103, and 123-140.

[0040] In some embodiments, the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 187-189. In some embodiments, the small prokaryotic ribosomal protein subunit S15 is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 181-183.

[0041] In some embodiments, the class 2 type V Cas effector and Tn7-type transposase complex is encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0042] In some aspects, the present disclosure provides an engineered nuclease system comprising: a nuclease comprising a RuvC domain, the nuclease being derived from an uncultured microorganism and being a Class 2 VK-type Cas effector, the Class 2 VK-type Cas effector comprising at least 80% identity with any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220; and an engineered guide RNA configured to form a complex with the nuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence.

[0043] In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least 80% identity to any one of SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 106, 107, 108, 5, 45-63, 68-75, 96-103, and 123-140.

[0044] In some aspects, the present disclosure provides a method for transposing a cargo nucleotide sequence into a target nucleic acid site, the method comprising introducing a system of the present disclosure into a cell.

[0045] In some aspects, the present disclosure provides a cell, which includes a system of the present disclosure. In some embodiments, the cell is a eukaryotic cell. 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 cell, HT1080, HepG2, Huh7, K562, primary cell or its derivative. In some embodiments, the cell is an engineered cell. In some embodiments, the cell is a stable cell.

[0046] For those skilled in the art, further aspects and advantages of the present disclosure will become apparent through the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other different embodiments, and its several details can be modified in various obvious aspects, all without departing from the present disclosure. Therefore, the drawings and description are to be regarded as illustrative rather than restrictive in nature. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The novel features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description setting forth illustrative embodiments in which the principles of the present disclosure are utilized, and in the accompanying drawings (also referred to herein as "Figures (Figure / FIG.)"):

[0048] Figure 1 Example organizations of different species and types of CRISPR / Cas loci are depicted.

[0049] Figure 2 Depicted is the architecture of the native 2 type II crRNA / tracrRNA pair shown for, e.g., Cas9 compared to a hybrid sgRNA in which the crRNA and tracrRNA are linked.

[0050] Figure 3 Two pathways found in Tn7 and Tn7-like elements are depicted.

[0051] Figures 4A-4B The genomic background of the V-type Tn7 CAST of the MG64 family is depicted. Figure 4A The MG64-1 CAST system is depicted including a CRISPR array (CRISPR repeats), a V-type nuclease, and three predicted transposase protein sequences. TracrRNA is predicted in the intergenic region between the CAST effector and the CRISPR array. Bottom: Multiple sequence alignment of the catalytic domain of the transposase TnsB. Catalytic residues are indicated by boxes. Figure 4B The two transposon ends predicted for the MG64-1 CAST system are depicted.

[0052] Figure 5 Depicted are the predicted structures of the corresponding sgRNAs for the CAST system described herein. Figure 5 Panel A (left) shows the predicted MG64-1 tracrRNA and crRNA duplex at the repeat-antirepeat stem. The loop was truncated and the tetraloop of GAAA was added to the stem-loop structure to generate Figure 5 Designed sgRNAs shown in panel B (right).

[0053] Figure 6 Depicted are the results of transposition reactions targeting a plasmid library consisting of NNNNNNNN 5' of the target spacer sequence. Reaction #1 shows the presence of the target library, reaction #2 shows the presence of the donor fragment in both transposition reactions, and reactions #3-5 show sg-specific PCR bands corresponding to the appropriate transposition reactions.

[0054] Fig. 7A -7D depicts the results of Sanger sequencing. Fig. 7A The Sanger sequencing of the donor target junction on the transposon LE in the left end (LE) transposition reaction closer to PAM is shown. The expected sequence is located at the top of the small figure, where the predicted transposition event is 61bp away from PAM. The top chromatogram is the sequencing result starting from the donor fragment. Clear signals can be seen on the right end until the donor / target junction (dashed line). This represents the mixing of the transposition product. The bottom chromatogram of the small figure is the sequencing from the target to the donor / target junction. The signal from the left side is to remove the signal until the junction. Figure 7B Shown is the Sanger sequencing of the donor target junction on the transposon right end (RE) in the product of the LE closer to PAM. The expected sequence is located at the top of the inset, where the predicted transposition event is 61bp away from PAM. The top chromatogram is the sequencing result starting from within the donor fragment. Clear signals can be seen on the left end until the donor / target junction (dashed line). Figure 7C is a close-up of the PAM library. Figure 7D is a SeqLogo analysis of NGS of events closer to the LE of the PAM, indicating a very strong preference for NGTN in the PAM motif.

[0055] Figure 8 A phylogenetic gene tree of Cas12k effector sequences is depicted. The tree is inferred from a multiple sequence alignment of 64 Cas12k sequences (orange and black branches) recovered here and 229 reference Cas12k sequences (grey branches) from public databases. The orange branches indicate Cas12k effectors that have been confirmed to be associated with CAST transposon components.

[0056] Fig. 9 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 repeat anti-repeat (RAR) 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)).

[0057] Fig. 10A and Fig. 10B Depicted is the secondary structure predicted by folding the CRISPR repeat+tracrRNA of the MG64 system.

[0058] Fig.11A The MG64-3 CRISPR locus is depicted. The tracrRNA is encoded upstream of the CRISPR array, while the transposon end is encoded downstream (inner black box). The sequence corresponding to part of the 3' CRISPR repeat sequence and part of the spacer are encoded within the transposon (outer frame). The self-matching spacer is encoded outside the transposon end.

[0059] Fig. 11B The tracrRNA sequence alignment of various CASTs provided herein is depicted. The alignment of tracrRNA sequences shows the conserved region. Specifically, the sequence "TGCTTTC" at sequence positioning 92-98 (top frame) may be important for sgRNA tertiary structure and non-continuous repeat-anti-repeat pairing with crRNA. The hairpin "CYCC (n6) GGRG" at positioning 265-278 (bottom frame) may be important for function, such as by positioning the downstream sequence of crRNA pairing.

[0060] Fig. 12A The predicted structure of the MG64-1 sgRNA is depicted.

[0061] Fig. 12B The predicted structure of the MG64-3 sgRNA is depicted.

[0062] Fig. 12C The predicted structure of the MG64-5 sgRNA is depicted.

[0063] Figures 13A-13C Depicted are PCR data demonstrating that MG64-1 is active against sgRNA v2-1. Using the described in vitro targeted integrase activity protocol, the effector protein and its TnsB, TnsC, and TniQ proteins were expressed in an in vitro transcription / translation system. After translation, the target DNA, cargo DNA, and sgRNA were added to the reaction buffer. Integration was determined by PCR across the target / donor junction. Fig.13A A diagram showing the possible orientations of the integrated donor DNA is depicted. PCR reactions 3, 4, 5 and 6 represent each integration junction product, depending on the orientation of the donor integration at the target site. Fig. 13B Gel images of PCR 4 (detection of RE junctions with donor) for transposition are depicted, showing: lane 1) apo (no sgRNA); lane 2) with sgRNA 1; and lane 3) with sgRNA v2-1. Fig. 13CGel images of PCR 5 (detection of LE junctions with donor) for transposition are depicted, showing: lane 1) apo (no sgRNA); lane 2) with sgRNA 1; and lane 3) with sgRNA v2-1.

[0064] Fig.14 PCR reaction 5 (LE close to PAM, top half of the graph) and PCR reaction 4 (RE far from PAM, bottom half of the graph) are depicted on the sequence of MG64-1 and plotted at a certain distance from the PAM. Analysis of the integration window showed that 95% of integrations occurring at the spacer PAM site were within a 10 bp window between 58 and 68 nucleotides from the PAM. The difference in integration distance between distal and proximal frequencies reflects integration site duplications - 3-5 base pair duplications are the result of interleaving of nuclease activity of the transposase upon integration.

[0065] Fig.15 Depicted are the results of a colony PCR screen for transposition efficiency. After incubation, 18 colony forming units (CFU) were visible on the plates; 8 on plate A (no IPTG, lane labeled A), and 10 on plate B (with 100 μM IPTG in the recovery, lane labeled B). All 18 were analyzed by colony PCR, which gave product bands (arrows) indicating successful transposition reactions.

[0066] Fig.16 Depicted are the sequencing results of the selected colony PCR products, which confirm that they represent transposition events because they span the junction between the LE and the PAM at the engineered target site located in the lacZ gene. The minimum LE sequence is represented in blue at the top of the screen (min LE), while the target and PAM are represented in gray. Some sequence variation was observed in the PCR products, but this variation is expected considering that insertions may occur at variable distances upstream of the PAM.

[0067] Fig.17 Depicted are the results of testing the 64-1 transposition activity of the engineered single guide. The black boxes are lanes not relevant to this experiment. Fig.17 Panel A depicts a gel image of PCR 4 of transposition (detection of RE junction with donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = sgRNA v1-1; lane 4 = sgRNA v1-2; lane 5 = sgRNAv1-3. Fig.17Panel B depicts a gel image of PCR 5 of transposition (detection of LE junctions 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. Fig.17 Panel C depicts a gel image of PCR 4 of transposition (detecting the RE junction with the donor): 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. Fig.17 Panel D depicts a gel image of PCR 5 of transposition (detecting LE junctions 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. Fig.17 Panel E depicts a gel image of PCR 4 of transposition (detection of RE junction with donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = sgRNA v1-5; lane 4 = skip; lane 5 = sgRNA v1-10. Fig.17 Panel F depicts a gel image of PCR 5 of transposition (detection of LE junctions with donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = sgRNA v1-5; lane 4 = skip; lane 5 = sgRNA v1-10. Fig.17 Panel G depicts a gel image of PCR 4 of transposition (detecting RE junctions with donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = sgRNAv1-17; lane 4 = sgRNA v1-18; lane 5 = skip; lane 6 = sgRNA v1-19; lane 7 = skip; lane 8 = sgRNA v1-20. Fig.17 Panel H depicts a gel image of PCR5 of transposition (detecting LE junctions with donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = sgRNAv1-17; lane 4 = sgRNA v1-18; lane 5 = skip; lane 6 = sgRNA v1-19; lane 7 = skip; lane 8 = sgRNA v1-20.

[0068] Fig.18 Depicted are the results of testing the 64-1 transposition activity of engineered LE and RE. Black boxes are lanes not relevant to this experiment. Fig.18 Panel A depicts a gel image of PCR 4 of transposition (detecting the RE junction 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. Fig.18 Panel B depicts a gel image of PCR 5 of transposition (detecting the LE junction 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. Fig.18 Panel C depicts a gel image of PCR 4 of transposition (detecting the RE junction with the donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = RE internal deletion 81 and 178 bp; lane 4 = skipped; lane 5 = RE internal deletion 81 and 196 bp; lane 6 = skipped; lane 7 = RE internal deletion 81 and 212 bp; lane 8 = skipped. Fig.18 Panel D depicts a gel image of PCR 5 of transposition (detecting the LE junction with the donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = RE internal deletion 81 and 178 bp; lane 4 = skipped; lane 5 = RE internal deletion 81 and 196 bp; lane 6 = skipped; lane 7 = RE internal deletion 81 and 212 bp; lane 8 = skipped. Fig.18 Panel E depicts a gel image of PCR 4 of transposition (detecting the RE junction with the donor): 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. Fig.18Panel F depicts a gel image of PCR 5 of transposition (detecting the LE junction with the donor): 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. Fig.18 Panel G depicts a gel image of PCR 6 of transposition (detecting the RE junction with the donor): lane 1 = apo (no sgRNA); lane 2 = holo (+ sgRNA); lane 3 = 0 bp overhang; lane 4 = 1 bp overhang; lane 5 = 2 bp overhang; lane 6 = 3 bp overhang; lane 7 = 5 bp overhang; lane 8 = 10 bp overhang.

[0069] Fig.19 Depicted are the results of testing the transposition activity of engineered CAST components with NLS. Black boxes are lanes not relevant to this experiment. Fig.19 Panel A depicts a gel image of PCR 4 of transposition (detecting the RE junction with the donor): lane 1 = apo (no sgRNA); lane 2 = holo (+ sgRNA); lane 3 = skip; lane 4 = skip; lane 5 = skip; lane 6 = NLS-TnsB; lane 7 = skip; lane 8 = TnsB-NLS. Fig.19 Panel B depicts a gel image of PCR 5 of transposition (detecting the LE junction with the donor): lane 1 = apo (no sgRNA); lane 2 = holo (+ sgRNA); lane 3 = skip; lane 4 = skip; lane 5 = skip; lane 6 = NLS-TnsB; lane 7 = skip; lane 8 = TnsB-NLS. Fig.19 Panel C depicts a gel image of PCR 4 of transposition (detecting the RE junction with the donor): 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. Fig.19 Panel D depicts a gel image of PCR 5 of transposition (detecting the LE junction with the donor): 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. Fig.19Panel E depicts a gel image of PCR 4 of transposition (detecting the RE junction with the donor): lane 1 = apo (no sgRNA); lane 2 = holo (+ sgRNA); lane 3 = skip; lane 4 = skip; lane 5 = NLS-Cas12k; lane 6 = Cas12k-NLS; lane 7 = NLS-TnsC; lane 8 = TnsC-NLS. Fig.19 Panel F depicts a gel image of PCR 5 of transposition (detecting the LE junction with the donor): lane 1 = apo (no sgRNA); lane 2 = holo (+ sgRNA); lane 3 = skip; lane 4 = skip; lane 5 = NLS-Cas12k; lane 6 = Cas12k-NLS; lane 7 = NLS-TnsC; lane 8 = TnsC-NLS. Fig.19 Panel G depicts a gel image of PCR 4 of transposition (detecting the RE junction 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. Fig.19 Panel H depicts a gel image of PCR 5 of transposition (detecting the LE junction 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. Fig.19 Panel I depicts a gel image of PCR 4 of transposition (detection of RE junction with donor): Lane 1 = apo (no sgRNA); Lane 2 = holo (+sgRNA); Lane 3 = Cas 2x NLSapo (no sgRNA); Lane 4 = Cas 2x NLS holo (+sgRNA). Fig.19 Panel J depicts a gel image of PCR 5 of transposition (detection of LE junction with donor): Lane 1 = apo (no sgRNA); Lane 2 = holo (+sgRNA); Lane 3 = Cas 2x NLS apo (no sgRNA); Lane 4 = Cas 2x NLS holo (+sgRNA).

[0070] Fig. 20Engineered CAST-NLS is depicted as a single kit. Unless otherwise described, all lanes have Cas12k-NLS and NLS-TniQ, TnsB, TnsC and sgRNA. Fig. 20 Panel A depicts a gel image of PCR 4 of transposition (detecting the RE junction with the donor): 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. Fig. 20 Panel B depicts a gel image of PCR 5 of transposition (detecting the LE junction with the donor): 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.

[0071] Fig.21 Depicted are the results of testing the transposition activity of Cas effector and TniQ protein fusions. Fig.21 Panel A depicts a gel image of PCR 4 (detection of RE junction with donor) of transposition: Lane 1 = apo (no sgRNA) and Cas-TniQ fusion; Lane 2 = holo (+ sgRNA) and Cas-TniQ fusion; Lane 3 = apo (no sgRNA) and TniQ-Cas fusion; Lane 4 = holo (+ sgRNA) and TniQ-Cas fusion. Fig.21 Panel B depicts a gel image of PCR 5 (detection of LE junctions with donor) of transposition: Lane 1 = apo (no sgRNA) with Cas-TniQ fusion; Lane 2 = holo (+sgRNA) with Cas-TniQ fusion; Lane 3 = apo (no sgRNA) with TniQ-Cas fusion; Lane 4 = holo (+sgRNA) with TniQ-Cas fusion. Fig.21Panel C depicts a gel image of PCR 4 of transposition (detecting the RE junction with the donor): lane 1 = apo (no sgRNA) with TniQ-Cas fusion; lane 2 = holo (+sgRNA) with TniQ-Cas fusion; lane 3 = holo Cas alone; lane 4 = apo (no sgRNA) with TniQ-48 linker-Cas fusion; lane 5 = holo (+sgRNA) with TniQ-48 linker-Cas fusion; lane 6 = apo (no sgRNA) with TniQ-68 linker-Cas fusion; lane 7 = holo (+sgRNA) with TniQ-68 linker-Cas fusion; lane 8 = holo (+sgRNA) with TniQ-72 linker-Cas fusion. Fig.21 Panel D depicts a gel image of PCR 5 of transposition (detecting LE junctions with donors): lane 1 = apo (no sgRNA) fused to TniQ-Cas; lane 2 = holo (+sgRNA) fused to TniQ-Cas; lane 3 = holo Cas alone; lane 4 = apo (no sgRNA) fused to TniQ-48 linker-Cas; lane 5 = holo (+sgRNA) fused to TniQ-48 linker-Cas; lane 6 = apo (no sgRNA) fused to TniQ-68 linker-Cas; lane 7 = holo (+sgRNA) fused to TniQ-68 linker-Cas; lane 8 = holo (+sgRNA) fused to TniQ-72 linker-Cas. Fig.21 Panel E depicts a gel image of PCR4 of transposition (detecting the RE junction with the donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = apo (no sgRNA) with NLS-TniQ-Cas-NLS fusion; lane 4 = holo (+sgRNA) with NLS-TniQ-Cas-NLS fusion; lane 5 = apo (no sgRNA) with NLS-TniQ-77 linker-Cas-NLS fusion; lane 6 = holo (+sgRNA) with NLS-TniQ-77 linker-Cas-NLS fusion. Fig.21Panel F depicts a gel image of PCR 5 of transposition (detecting the LE junction with the donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = apo (no sgRNA) with NLS-TniQ-Cas-NLS fusion; lane 4 = holo (+sgRNA) with NLS-TniQ-Cas-NLS fusion; lane 5 = apo (no sgRNA) with NLS-TniQ-77 linker-Cas-NLS fusion; lane 6 = holo (+sgRNA) with NLS-TniQ-77 linker-Cas-NLS fusion. Fig.21 Panel G depicts a gel image of PCR4 of transposition (detecting the RE junction with the 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-TniQ holo (+sgRNA). Fig.21 Panel H depicts a gel image of PCR 5 of transposition (detecting the LE junction with the 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-TniQ holo (+sgRNA).

[0072] Fig. 22 Depicted are the expression of TnsB and TnsC in human cells followed by cell fractionation and the results of in vitro transposition reactions. Fig. 22Panel A depicts a gel image of PCR 4 of transposition (detecting RE junctions with donors): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = holo (+sgRNA) and untreated (no TnsB) cytoplasm; lane 4 = holo (+sgRNA) and untreated nucleoplasm; lane 5 = holo (+sgRNA) and NLS-TnsB cell cytoplasm; lane 6 = holo (+sgRNA) and NLS-TnsB cell nucleoplasm; lane 7 = holo (+sgRNA) and TnsB-NLS cell cytoplasm; lane 8 = holo (+sgRNA) and TnsB-NLS cell nucleoplasm; lane 9 = holo (+sgRNA) and NLS-TniQ cell cytoplasm; lane 10 = holo (+sgRNA) and NLS-TniQ cell nucleoplasm. Fig. 22 Panel B depicts a gel image of PCR 5 of transposition (detecting LE junctions with the donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = holo (+sgRNA) and untreated (no TnsB) cytoplasm; lane 4 = holo (+sgRNA) and untreated nucleoplasm; lane 5 = holo (+sgRNA) and NLS-TnsB cell cytoplasm; lane 6 = holo (+sgRNA) and NLS-TnsB cell nucleoplasm; lane 7 = holo (+sgRNA) and TnsB-NLS cell cytoplasm; lane 8 = holo (+sgRNA) and TnsB-NLS cell nucleoplasm; lane 9 = holo (+sgRNA) and NLS-TniQ cell cytoplasm; lane 10 = holo (+sgRNA) and NLS-TniQ cell nucleoplasm. Fig. 22 Panel C depicts a gel image of PCR 4 of transposition (detecting the RE junction with the donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = holo (+sgRNA) without TnsC; lane 4 = holo (+sgRNA) and untreated (no TnsC) cytoplasm; lane 5 = holo (+sgRNA) and untreated nucleoplasm; lane 6 = holo (+sgRNA) and NLS-HA-TnsC cell cytoplasm; lane 7 = holo (+sgRNA) and NLS-HA-TnsC cell nucleoplasm; lane 8 = holo (+sgRNA) and TnsC-NLS cell cytoplasm; lane 9 = holo (+sgRNA) and TnsC-NLS cell nucleoplasm. Fig. 22Panel D depicts a gel image of PCR 5 of transposition (detecting the LE junction with the donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = holo (+sgRNA) without TnsC; lane 4 = holo (+sgRNA) and untreated (no TnsC) cytoplasm; lane 5 = holo (+sgRNA) and untreated nucleoplasm; lane 6 = holo (+sgRNA) and NLS-HA-TnsC cell cytoplasm; lane 7 = holo (+sgRNA) and NLS-HA-TnsC cell nucleoplasm; lane 8 = holo (+sgRNA) and TnsC-NLS cell cytoplasm; lane 9 = holo (+sgRNA) and TnsC-NLS cell nucleoplasm. Fig. 22 Panel E depicts a gel image of PCR 4 of transposition (detection of RE junction with donor): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = apo (no sgRNA) NLS-TnsB-IRES-NLS-TnsC cytoplasmic; Lane 4 = holo (+ sgRNA) NLS-TnsB-IRES-NLS-TnsC cytoplasmic; Lane 5 = apo (no sgRNA) NLS-TnsB-IRES-NLS-TnsC nucleoplasmic; 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 (no sgRNA) TnsB-NLS-IRES-NLS-TnsC nucleoplasm; lane 10 = holo (+sgRNA) TnsB-NLS-IRES-NLS-TnsC nucleoplasm. Fig. 22 Panel F depicts a gel image of PCR 5 of transposition (detection of LE junction with donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = apo (no sgRNA) NLS-TnsB-IRES-NLS-TnsC cytoplasmic; lane 4 = holo (+sgRNA) NLS-TnsB-IRES-NLS-TnsC cytoplasmic; lane 5 = apo (no sgRNA) NLS-TnsB-IRES-NLS-TnsC nucleoplasmic; lane 6 = holo (+sgRNA) NLS-TnsB-IRES-NLS-TnsC nucleoplasmic; lane 7 = apo (no sgRNA) TnsB-NLS-IRES-NLS-TnsC cytoplasmic; lane 8 = holo (+sgRNA)

[0073] TnsB-NLS-IRES-NLS-TnsC cytoplasm; Lane 9 = apo (no sgRNA)

[0074] TnsB-NLS-IRES-NLS-TnsC nucleoplasm; Lane 10 = holo (+ sgRNA)

[0075] TnsB-NLS-IRES-NLS-TnsC nucleoplasm.

[0076] Fig.23 Depicted are the results of expression of Cas12k and TniQ-linked constructs in human cells followed by in vitro transposition assays. Fig.23 Panel A depicts a gel image of PCR 5 of transposition (detection of LE junctions 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 =

[0077] Cas-NLS-P2A-NLS-TniQ holo (+sgRNA) nucleoplasm; Lane 8 =

[0078] Cas-NLS-P2A-NLS-TniQ holo(+sgRNA) nucleoplasm+additional sgRNA. Fig.23 Panel B depicts a gel image of PCR 4 of transposition (detection of RE junctions with donors): lane 1 = apo (no sgRNA); lane 2 = holo (+ sgRNA); lane 3 = apo (no sgRNA) Cas-NLS-P2A-NLS-TniQ cytoplasmic; lane 4 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ cytoplasmic; lane 5 = apo (no sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasmic; lane 6 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasmic; lane 7 = holo (+ sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasmic + additional holo Cas-NLS; Lane 8 = holo (+sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm + NLS-TniQ. Fig.23Panel C depicts a gel image of PCR 5 of transposition (detection of LE junction with donor): Lane 1 = apo (no sgRNA); Lane 2 = holo (+ sgRNA); Lane 3 = apo (no sgRNA)Cas-NLS-P2A-NLS-TniQ cytoplasmic; Lane 4 = holo (+ sgRNA)

[0079] 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 = holo (+sgRNA) Cas-NLS-P2A-NLS-TniQ nucleoplasm + NLS-TniQ. Fig.23 Panel D depicts a gel image of PCR 4 of transposition (detecting the RE junction with the donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = apo (no sgRNA) NLS-TniQ-Cas-NLS cytoplasmic; lane 4 = holo (+sgRNA) NLS-TniQ-Cas-NLS cytoplasmic; 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. Fig.23 Panel E depicts a gel image of PCR 5 of transposition (detecting the LE junction with the donor): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = apo (no sgRNA) NLS-TniQ-Cas-NLS cytoplasmic; lane 4 = holo (+sgRNA) NLS-TniQ-Cas-NLS cytoplasmic; 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. Fig.23Panel F depicts a gel image of PCR 4 of transposition (detection of RE junction with donor): lane 1 = apo (no sgRNA); lane 2 = holo (+ sgRNA); lane 3 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ cytoplasmic; lane 4 = holo (+ sgRNA) Cas-NLS-IRES-NLS-TniQ cytoplasmic; lane 5 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasmic; lane 6 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasmic + additional PURExpress; lane 7 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasmic + additional PURExpress; lane 8 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasmic 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. Fig.23Panel G depicts a gel image of PCR 5 of transposition (detection of LE junctions with donors): lane 1 = apo (no sgRNA); lane 2 = holo (+sgRNA); lane 3 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ cytoplasmic; lane 4 = holo (+sgRNA) Cas-NLS-IRES-NLS-TniQ cytoplasmic; lane 5 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasmic; lane 6 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasmic + additional PURExpress; lane 7 = apo (no sgRNA) Cas-NLS-IRES-NLS-TniQ nucleoplasmic + additional PURExpress; 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.

[0080] Fig.24 Electrophoretic mobility shift assay (EMSA) results of 64-1TnsB and its LE DNA sequence are depicted. EMSA results confirmed binding and TnsB recognition. TnsB protein was expressed in an in vitro transcription / translation system, incubated with FAM-labeled DNA containing the LE sequence, and then separated on a native 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-labeled DNA only. Lane 2: FAM DNA plus in vitro transcription / translation system (no TnsB protein). Lane 3: FAM DNA plus TnsB.

[0081] Figures 25A-25B Cas12k effector diversity is profiled. Fig.25AThe Cas12k CAST genomic context is depicted. The transposon is characterized by: terminal inverted repeats (TIR, light orange bars), Tn7-like transposon genes (colored arrows), death effector Cas12k (orange arrows), tracrRNA (pink half arrows), and CRISPR arrays. "TAAA" target site repeats (TDS) were observed to flank TIR. Middle panel: MG64-1 noncoding region illustration, showing tracrRNA (a pseudo-repeat sequence and self-targeting spacer), CRISPR array, and transposon left end TIR. Bottom panel: Multiple alignments of pseudo-repeat sequences and self-targeting spacers in a group of CAST homologs. Fig.25B Depicted is an unrooted phylogenetic tree of Cas12k effectors. The Cas12k effectors recovered in this study are shown in orange (confirmed transposons in the genome) and black branches, while the reference Cas12k sequences are shown in gray. The reference sequences ShCas12k and AcCas12k are shown with red arrows.

[0082] Figures 26A-26B Depicts the right end of CAST ( Fig.26A ) and the left end ( Fig.26B ). The transposon end inverted motif "TGTNNA" is highlighted with a box.

[0083] Fig. 27 Depicted is the alignment of the Cas12k CAST tracrRNA sequence, showing regions of sequence and structural conservation. Specifically, the sequence "TGCTTTC" at sequence location 88-92 may be important for sgRNA tertiary structure and non-continuous repeat-anti-repeat pairing with crRNA. The hairpin "CYCC (n6) GGRG" at location 279-294 may be important for function and may locate the downstream sequence of crRNA pairing.

[0084] Fig.28 Single guide RNA folding of active MG64-1, MG64-2, and MG64-6 CAST systems is depicted. Also shown is an active engineered sgRNA for MG64-1.

[0085] FIG. 29 depicts in vitro screening of CAST transposition using a PAM library. Fig.29A The screening setup of the in vitro PAM assay is depicted. Fig.29B A schematic diagram of junction PCR for detection of transposition products is depicted.

[0086] Fig. 30A Transposition junctions of MG64-1 CAST (left lane) and MG64-6 CAST (right lane) amplified by PCR are depicted.

[0087] Fig. 30B SeqLogo representation of detected PAMs of MG64-1 is depicted (top).

[0088] Fig. 30C Depicted are the integrated frequencies plotted by distance at proximal and distal distances of MG64-1.

[0089] Fig.31 Depicted is the single guide RNA engineering of 64-1. Deletion of a region of approximately 130 bp to 190 bp (green and cyan segments of the structure) resulted in a sgRNA that directed a strong transposition reaction (green bar on the heat map).

[0090] Fig.32 Cross-reactivity of MG64-2 sgRNA to MG64-1 and PAM for the combination of MG64-2 sgRNA plus MG64-1 effector is depicted.

[0091] Fig.33 Single guide RNA truncations in the coding DNA are depicted for MG64-2 sgRNA in the sequence view and secondary structure prediction model. Deleted regions and truncations are shown as bars (del1, del2, del3, del4, del5, and del6) in the sequence view. Deleted regions in ovals in the secondary structure prediction model indicate the truncations tested (deletions 1, 2, 3, 4 on pseudoknots, 5, and 6).

[0092] Fig.34 Data demonstrating that the engineered MG64-2 sgRNA is active on the MG64-1 CAST system are depicted. PCR reactions represent each possible integration junction or negative control (Figure 29, panel B). Successful integration products are highlighted with arrows. The boxed lanes are not relevant to this experiment.

[0093] Fig.35 MG64-2 sgRNA split guide design depicted. The sgRNA fragments were synthesized separately and then re-annealed before testing in transposition experiments.

[0094] Fig.36 Data demonstrating that the split MG64-2 sgRNA is active on the MG64-1 CAST system are depicted. PCR reactions represent each possible integration junction or negative control (Figure 29, panel B). Successful integration products are highlighted with arrows. Boxed lanes are not relevant to this experiment.

[0095] Fig.37 Depicted are data demonstrating that LE and RE minimization maintains the transposition activity of the system.

[0096] Figures 38A-38CThe results of E. coli integration with MG64-1 are depicted. Fig.38A Depicted is a schematic representation of the introduction of the CAST system into E. coli. Fig.38B Depicted are NGS data showing editing efficiency greater than 80%. Fig.38C Depicted is an off-target analysis showing no off-target integrations greater than 1% of all transposition events added were detected.

[0097] Fig.39 The local insertion rates of various endogenous loci of the E. coli genome are depicted.

[0098] Figures 40A-40B Results of multilocus targeting are depicted. Fig.40A The corresponding local insertion frequencies at endogenous and engineered loci are depicted. Fig.40B The relative insertion frequencies of on-target insertions at the endogenous locus, on-target insertions at the engineered locus, and off-target insertions are depicted. Integrations at both loci together account for more than 95% of all integrations occurring on the genome.

[0099] Fig.41 Depicted are Sanger sequencing data of the integrated PCR products, demonstrating that MG64-1 is active in vitro. The reaction is that of RE donor-target product, and sequencing stops when the junction occurs matching the donor DNA (dark bar below the sequencing peak).

[0100] Fig.42A A schematic representation of serial dilutions of target DNA for in vitro transposition experiments is shown. CAST components were expressed with PureExpress and added to reactions with in vitro transcribed sgRNA and donor plasmid. Target plasmid DNA was added at decreasing concentrations and tested in transposition experiments. When the minimum amount of target DNA was determined, the transposition reaction was assayed by adding increasing amounts of human genomic DNA.

[0101] Fig.42B A schematic representation of the PCR amplification of the transposition reaction is shown. The 8N PAM plasmid library (8N-target, Rxn#1) was targeted with the CAST system to integrate donor DNA (Rxn#2). After successful integration, a junction PCR reaction was performed with primers to amplify four putative integration reactions (Rxn#3, #4, #5, and #6) based on the orientation of the cargo integration.

[0102] Fig.42C PCR reaction products from an in vitro transposition assay performed with serial dilutions of target plasmid DNA are shown. Target, donor, and reactions #3, #4, #5, and #6 correspond to Fig.42B PCR integration products are shown.

[0103] Fig.42D PCR reaction products from an in vitro transposition assay with a fixed amount of target plasmid DNA (0.5 ng) are shown, while increasing amounts of human genomic DNA are added to increase the search space. Target, donor, and reactions #3, #4, #5, and #6 correspond to Fig.42B PCR integration products are shown.

[0104] Fig.43A A schematic diagram of the transposition reaction across high copy elements is shown. When measured with CAST protein and sgRNA targeting one of multiple array targets, the target PCR product spans the wild-type target element. Integration can occur in the forward orientation, reverse orientation, or in both. The forward transposition product is measured by amplifying the junction PCR covering the LE of the donor DNA to the region at the 5' end of the target site (forward PCR). The reverse junction reaction measures the region covering the LE of the donor DNA to the 3' end of the target element (inverse PCR).

[0105] Fig.43B PCR reaction products from an in vitro transposition assay at 15 target sites (guides) of the LINE1 3' element in human genomic DNA are shown. Targets and reactions Forward PCR and reverse PCR correspond to Fig.43A PCR integration products are shown.

[0106] Fig.43C PCR reaction products from an in vitro transposition assay at 15 target sites (guides) of the SVA element in human genomic DNA are shown. Targets and reactions Forward PCR and reverse PCR correspond to Fig.43A PCR integration products are shown. Bands are highlighted with arrows indicating successful targeted integration.

[0107] Fig.43D PCR reaction products from an in vitro transposition assay at 15 target sites (guides) of HERV elements in human genomic DNA are shown. Targets and reactions Forward PCR and reverse PCR correspond to Fig.43A PCR integration products are shown. Bands are highlighted with arrows indicating successful targeted integration.

[0108] Fig.43E Sanger sequencing of forward PCR integration products at multiple target sites of the LINE1 3' element is shown. Where integration occurs, the sequencing trace stops matching the donor DNA (grey vertical bars).

[0109] Fig.43FSanger sequencing of inverse PCR integration products at multiple target sites of the LINE1 3' element is shown. Where integration occurs, the sequencing trace stops matching the target DNA (grey vertical bars).

[0110] Figure 43G Shown is Sanger sequencing of the forward PCR integration product at SVA target site 3. Where integration occurred, the sequencing trace stops matching the donor DNA (grey vertical bars).

[0111] Figure 43H Shown is Sanger sequencing of the forward PCR integration product at HERV target site 5. Where integration occurred, the sequencing trace stops matching the donor DNA (grey vertical bars).

[0112] Fig.44 PCR reaction products from an in vitro transposition assay at LINE1 target sites 12 and 15 in human genomic DNA with functional domains are shown. Targets and reactions Forward PCR and reverse PCR correspond to Fig.42A PCR integration products are shown. Bands are highlighted with arrows indicating successful targeted integration.

[0113] Figures 45A-45B In vitro transposition experiments using CAST, S15, NLS-S15, and S15-NLS expressed by a eukaryotic transcription / translation reaction are shown. Fig.45A In vitro transposition reactions with MG64-1 CAST and S15 are shown. CAST components expressed from wheat germ extract promoted transposition without the addition of S15, albeit at a lower rate (faint bands highlighted with arrows). Addition of PURExpress reagent (used PUREx) increased transposition efficiency, as shown by the intensity of the band at Rxn#5 (PURExpress reagent contains S15). Independent addition of S15 and S15-NLS translated from wheat germ extract reactions increased the transposition efficiency of MG64-1 in vitro (strong bands highlighted with arrows) compared to other conditions tested. Fig.45B In vitro reactions of transposition with NLS-S15 configurations are shown. Addition of PURExpress reagent increased in vitro transposition (lane 3) compared to the CAST component only condition (lane 2). NLS-S15 configuration did not improve transposition (lanes 4-5). Box Rxn#5 indicates the expected band if transposition activity is detected.

[0114] Figures 46A-46H A schematic diagram of the fusion plasmid used for intracellular transposition is shown. Fig.46A : Two targeting complex plasmids and one donor plasmid were assembled for the high copy element Line1, targets 8, 12 and 15, and SVA target 3. Fig.46B Intracellular transposition to high copy elements with H1core-TniQ or HMGN1-TniQ is shown at LINE1 targets 8, 12, 15 and SVA target 3. Arrows indicate transposition junction reactions amplified in the forward (forward PCR) or reverse (inverse PCR) orientation of transposition. Mock controls represent reactions without targeting or donor plasmids. Fig.46C Sanger sequencing of forward PCR of PCR integration product at LINE1 3' target site 8 is shown. Integration is mediated by MG64-1 and NLS-H1core-TniQ fusion. Where integration occurs, the sequencing trace stops matching the donor DNA (grey vertical bars). Fig.46D Sanger sequencing of forward PCR of PCR integration product at LINE1 3' target site 8 is shown. Integration is mediated by MG64-1 and NLS-HMGN1-TniQ fusion. Where integration occurs, the sequencing trace stops matching the donor DNA (grey vertical bars). Fig.46E Sanger sequencing of inverse PCR of the PCR integration product at LINE1 3' target site 12 is shown. Integration is mediated by MG64-1 and the NLS-H1core-TniQ fusion. Where integration occurs, the sequencing trace stops matching the donor DNA (grey vertical bars). Fig.46F Sanger sequencing of inverse PCR of PCR integration products at LINE1 3' target site 12 is shown. Integration is mediated by MG64-1 and NLS-HMGN1-TniQ fusion. Where integration occurs, the sequencing trace stops matching the donor DNA (grey vertical bars). Figure 46G Sanger sequencing of the forward PCR of the PCR integration product at LINE1 3' target site 15 is shown. Integration is mediated by MG64-1 and the NLS-H1core-TniQ fusion. Where integration occurs, the sequencing trace stops matching the donor DNA (grey vertical bars). Figure 46H Sanger sequencing of the forward PCR of the PCR integration product at LINE1 3' target site 15 is shown. Integration is mediated by MG64-1 and the NLS-HMGN1-TniQ fusion. Where integration occurs, the sequencing trace stops matching the donor DNA (grey vertical bars).

[0115] Figures 47A-47D Depicted is immunofluorescence staining for localization of Cas12k CAST components in human cells. Fig.47A: Top 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 labeled with HA tag. Anti-HA antibody was used for protein detection. DAPI was used to stain DNA (nucleus). Fig.47B : Top and bottom rows: Detection of TniQ localization. Images show that MG64-1 TniQ is localized in the nucleus of mammalian cells. CAST protein was labeled with HA tag. Anti-HA antibody was used for protein detection. DAPI was used to stain DNA (nucleus). Fig.47C : All rows: Detection of co-localization of TnsC with TniQ. The images show that while some TnsC may remain in the cytoplasm, it is now co-localized with TniQ in the nucleus. CAST protein was labeled with HA tag. Anti-HA antibody was used for protein detection. DAPI was used to stain DNA (nucleus). Fig.47D : Two rows: Cas12k, TnsB, TnsC and TniQ co-delivered to HEK293T cells are localized in the nucleus. CAST protein is labeled with HA tag. Anti-HA antibody is used for protein detection. DAPI is used to stain DNA (nucleus).

[0116] Figures 48A-48B In vitro screening of MG64-1 Cas12k CAST transposition is depicted. Fig.48A : Diagram of constructs used for MG64-1 holo complex purification. Fig.48B : Schematic diagram of junction PCR for detection of transposition products. The target substrate with a 5' PAM followed by a protospacer (target, Rxn#1) was targeted with the CAST system to integrate cargo DNA (Rxn#2). After successful integration, junction PCR reactions were performed with primers to amplify four putative integration reactions based on the orientation of cargo integration. Figures 48C-48D MG64-1 protein purification is depicted. Fig.48C : Fractions collected during the 2-L scale purification of the MG64-1 holo complex were run on a stain-free denaturing PAGE gel. Fig.48D : Size Exclusion Chromatography (SEC) Chromatogram of MG64-1 holo complex. The peak centered at 29.3 mL (Peak 1) was used for in vitro activity assay.

[0117] Fig.49ADepicted are in vitro transposition of holo complexes recovered with peak 1 supplemented with components expressed by TnT. Lane L) ladder; Lane 1) TnT expressed CAST components apo condition (-sgRNA); Lane 2) TnT expressed CAST components holo condition (+sgRNA); Lane 3) purified peak 1 supplemented with TnT CAST components without additional supplementation with Cas12k (-TnTCas12k); Lane 4) purified peak 1 supplemented with TnT CAST components without additional supplementation with TnsC (-TnT TnsC); Lane 5) peak 1 supplemented with TnT CAST components without additional supplementation with TniQ (-TnT TniQ); Lane 6) peak 1 supplemented with TnT CAST components without additional supplementation with S15 (-TnT S15). Fig.49B Sanger sequencing of lanes 3, 4, 5, and 6 in the pdonor and target directions from the amplified LE to the PAM target-donor junction is depicted. The vertical line depicts the transposition junction of MG64-1 predicted in the reference sequence. The degradation of the signal from either direction is caused by the large amount of signal reflected in the PCR amplification.

[0118] Fig.50 Depicted is the identification of ribosomal protein S15 homologs in Cyanobacteria genomic fragments. Candidate sequences from the same sample as recovery MG64-1 are highlighted with black solid circles. The reference S15 from Escherichia coli is indicated with an arrow.

[0119] Brief Description of Sequence Listing

[0120] The sequence listing submitted herewith provides exemplary polynucleotide and polypeptide sequences for use in the methods, compositions and systems according to the present disclosure.Below is an exemplary description of the sequences therein.

[0121] MG64

[0122] SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85 and 220 show the full-length peptide sequences of the MG64 Cas effectors.

[0123] SEQ ID NOs: 2-4, 13-15, 17-19, 65-67 and 109-111 show peptide sequences of MG64 transposase proteins, which may include a recombinase complex associated with an MG64 Cas effector.

[0124] SEQ ID NOs: 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.

[0125] SEQ ID NOs:7 and 34-35 show the nucleotide sequence of the MG64 target CRISPR repeat sequence.

[0126] SEQ ID NOs: 106-108, 112-118 and 221 show the nucleotide sequences of MG64 crRNA.

[0127] SEQ ID NOs: 8, 10, 39-44, 77, 79 and 93 show the nucleotide sequences of the right transposase recognition sequences associated with the MG64 system.

[0128] SEQ ID NOs: 9, 11, 36-38, 76 and 78 show the nucleotide sequences of the left-sided transposase recognition sequences associated with the MG64 system.

[0129] SEQ ID NO:31 shows the PAM sequence associated with the MG64 Cas effector described herein.

[0130] SEQ ID NOs: 45-63, 68-75, 96-103 and 123-140 show the nucleotide sequences of single guide RNAs engineered to function with the MG64 Cas effector.

[0131] SEQ ID NO: 208 shows the nucleotide sequence of the MG64 expression construct.

[0132] MG190

[0133] SEQ ID NOs: 209-219 show the full-length peptide sequences of MG190 ribosomal protein S15 homologs.

[0134] Other sequences

[0135] SEQ ID NOs: 86-87 and 192-207 show the peptide sequences of nuclear localization signals.

[0136] SEQ ID NOs: 88-89 show the peptide sequences of the linkers.

[0137] SEQ ID NOs:90-92 show the peptide sequences of the epitope tags.

[0138] SEQ ID NOs: 141-143 show genomic target sequences.

[0139] SEQ ID NOs: 144-180 show targeting guide sequences.

[0140] SEQ ID NOs: 181-183 show the nucleic acid sequences of the S15 fusion proteins.

[0141] SEQ ID NO: 184 shows the donor construct.

[0142] SEQ ID NO: 185 shows the MG64-1 sgRNA sequence.

[0143] SEQ ID NO: 186 shows the linker sequence.

[0144] SEQ ID NOs: 187-189 show the amino acid sequences of the S15 fusion proteins.

[0145] SEQ ID NOs: 190-191 show promoter sequences. DETAILED DESCRIPTION

[0146] Although 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 may be envisioned by 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.

[0147] Unless otherwise indicated, the practice of certain methods disclosed herein employs techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA. See, e.g., Sambrook and Green et al., Molecular Cloning: A Laboratory Manual, 4th edition (2012); the book series Current Protocols in Molecular Biology (FM Ausubel et al., eds.); the book series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th edition (RI Freshney, ed., (2010)).

[0148] As used herein, the singular forms "a / an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, 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".

[0149] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one or more than one standard deviation, as practiced in the art. Alternatively, "about" can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0150] As used herein, "cell" refers to a biological cell. A cell can be the basic structural, functional and / or biological unit of a living organism. A cell can be derived from any organism having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants (e.g., cells from cultivated crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugar cane, pumpkin, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, lycopodiella, hornwort, liverwort, mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, C. agardh Sargassum pampatens C. Agardh, etc.), algae (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. Sometimes, the cell is not derived from a natural organism (e.g., the cell can be synthetically manufactured, sometimes referred to as an artificial cell).

[0151] As used herein, the term "nucleotide" refers to a base-sugar-phosphate combination. Nucleotide can include synthetic nucleotides. Nucleotide can include synthetic nucleotide analogs. Nucleotide can be a monomer unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphates, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP or its derivatives. Such derivatives can include, for example, [αS] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that impart nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide can refer to dideoxyribonucleoside triphosphates (ddNTP) and its derivatives. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to: ddATP, ddCTP, ddGTP, ddITP and ddTTP. Nucleotide can be unlabeled or detectably labeled, such as using a portion comprising an optically detectable portion (e.g., fluorophore). It can also be labeled with quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. The fluorescent labeling of nucleotides can include, but is 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-carboxyl-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo) benzoic acid (DABCYL), waterfall blue, Oregon green, Texas red, cyanine, and 5-(2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides can 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, Calif.; FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink FluorX-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 fluorescein-15-dATP, available from Molecular Probes, Inc., Eugene, Oregon. Probes, Eugene, Oreg.), BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade 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. Nucleotides can also be labeled or indicated by chemical modification. The chemically modified single nucleotide may be a biotin-dNTP.Some non-limiting examples of biotinylated dNTPs can 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).

[0152] The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides or their analogs, in single-stranded, double-stranded or multi-stranded form. The polynucleotide can be exogenous or endogenous to the cell. The polynucleotide can be present in a cell-free environment. The polynucleotide can be a gene or a fragment thereof. The polynucleotide can be DNA. The polynucleotide can be RNA. The polynucleotide can have any three-dimensional structure and can perform any function. In a polynucleotide, when T is mentioned, T means U (uracil) in RNA and T (thymine) in DNA. The polynucleotide can include one or more analogs (e.g., altered backbones, sugars or nucleobases). If present, modifications to the nucleotide structure can be imparted before or after assembling the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, heterologous nucleic acids, morpholinos, locked nucleic acids, glycerol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, braided nucleotides, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, multiple loci (one locus) defined according to linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes and primers. Nucleotide sequences may be interspersed with non-nucleotide components.

[0153] The term "transfection" or "transfected" refers to the introduction of nucleic acid into cells by non-viral or viral-based methods. The nucleic acid molecule can be a gene sequence encoding a complete protein or a functional portion thereof. See, for example, Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0154] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues connected by a peptide bond. This term does not indicate the specific length of the polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally present. The term is applicable to naturally occurring amino acid polymers and amino acid polymers including at least one modified amino acid. In some cases, the polymer may be interspersed with non-amino acids. The term encompasses amino acid chains of any length, including full-length proteins and proteins with or without secondary and / or tertiary structures (e.g., domains). The term also encompasses amino acid polymers that have been modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other operations, such as conjugation with a labeling component. As used herein, the terms "amino acid" and "amino acids" refer to natural and non-natural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids may include natural and non-natural amino acids that have been chemically modified to include groups or chemical moieties that are not naturally present on amino acids. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" encompasses D-amino acids and L-amino acids.

[0155] As used herein, "non-natural" may refer to nucleic acids or polypeptide sequences not found in natural nucleic acids or proteins. Non-natural may refer to affinity tags. Non-natural may refer to fusions. Non-natural may refer to naturally occurring nucleic acids or polypeptide sequences including mutations, insertions and / or deletions. Non-natural sequences may exhibit and / or encode activities (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) that may also be exhibited by nucleic acids and / or polypeptide sequences fused to non-natural sequences. Non-natural nucleic acids or polypeptide sequences may be connected to naturally occurring nucleic acids or polypeptide sequences (or variants thereof) by genetic engineering to produce chimeric nucleic acids and / or polypeptide sequences encoding chimeric nucleic acids and / or polypeptides.

[0156] As used herein, the term "promoter" refers to a regulatory DNA region that controls polynucleotide (e.g., gene) transcription or expression and can be located adjacent to or overlaps with a nucleotide or nucleotide region that initiates RNA transcription. A promoter may contain a specific DNA sequence that binds a protein factor (commonly referred to as a transcription factor) that promotes the binding of RNA polymerase to DNA, thereby causing gene transcription. 'Basic promoter', also referred to as 'core promoter', may refer to a promoter containing all the basic essential elements that promote the transcriptional expression of an operably linked polynucleotide. Eukaryotic basal promoters typically (although not necessarily) contain a TATA box and / or a CAAT box. In certain embodiments, different promoters direct the expression of genes in different tissues or cell types, or at different developmental stages, or in response to different environmental or physiological conditions or inducer molecules. Promoters that make genes expressed in most cell types at most times are generally referred to as "constitutive promoters". Promoters that make genes expressed in specific cells and tissue types are generally referred to as "cell-specific promoters" or "tissue-specific promoters", respectively. Promoters that make genes expressed at specific stages of development or cell differentiation are generally referred to as "developmental-specific promoters" or "cell differentiation-specific promoters". Promoters that are induced and result in gene expression following exposure or treatment of cells with an agent, biomolecule, chemical, ligand, light, etc. that induces the promoter are generally referred to as "inducible promoters" or "regulatable promoters." It is further recognized that in some embodiments, promoter activity is identical for DNA fragments of different lengths, as the exact boundaries of regulatory sequences have not been completely defined in most cases.

[0157] As used herein, the term "expression" refers to the process of transcribing a nucleic acid sequence or polynucleotide from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

[0158] As used herein, "operably connected", "operably connected", "operably connected" or its grammatical equivalent refers to genetic elements, such as the arrangement of promoters, enhancers, polyadenylation sequences, etc., wherein the operation (for example, movement or activation) of the first genetic element has some influence on the second genetic element. The influence on the second genetic element can be but need not be the same type as the operation of the first genetic element. For example, if the movement of the first element causes the activation of the second element, the two genetic elements are operably connected. For example, if the regulatory element helps to start the transcription of the coding sequence, the regulatory element that can include promoters and / or enhancer sequences is operably connected to the coding region. As long as this functional relationship is maintained, there will be an insertion residue between the regulatory element and the coding region.

[0159] As used herein, "vector" refers to a macromolecule or macromolecular association that includes or associates with a polynucleotide and can be used to mediate the delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. Vectors typically include genetic elements, such as regulatory elements, that are operably linked to a gene to promote expression of the gene in a target.

[0160] As used herein, "expression cassette" and "nucleic acid cassette" are used interchangeably to refer to a combination of nucleic acid sequences or elements that are expressed together or operably linked for expression. In some cases, an expression cassette refers to a combination of a regulatory element and one or more genes to which it is operably linked for expression.

[0161] A "functional fragment" of a DNA or protein sequence refers to a fragment that retains a biological activity (function or structure) substantially similar to that of the full-length DNA or protein sequence. The biological activity of a DNA sequence may be its ability to affect expression in a manner attributed to the full-length sequence.

[0162] The terms "engineered," "synthetic," and "artificial" are used interchangeably herein to refer to objects that are modified by human intervention. For example, the terms may refer to a non-naturally occurring polynucleotide or polypeptide. An engineered peptide may have, but need not have, low sequence identity (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) with a naturally occurring human protein. For example, the VPR and VP64 domains are synthetic transactivation domains. According to non-limiting examples: a nucleic acid may be modified by changing its sequence to a sequence that does not exist in nature; a nucleic acid may be modified by connecting it to a nucleic acid that is not associated with it in nature, so that the connection product has a function that does not exist in the original nucleic acid; an engineered nucleic acid may be synthesized in vitro with a sequence that does not exist in nature; a protein may be modified by changing its amino acid sequence to a sequence that does not exist in nature; an engineered protein may acquire new functions or properties. An "engineered" system includes at least one engineered component.

[0163] The term "tracrRNA" or "tracr sequence" means trans-activating CRISPR RNA. TracrRNA interacts with CRISPR (cr) RNA to form a guide nucleic acid (e.g., guide RNA or gRNA) that can hybridize with a target nucleic acid and thereby guide the relevant nuclease to the target nucleic acid. If tracrRNA is engineered, it may have about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% sequence identity and / or sequence similarity with a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes (S.pyogenes), Staphylococcus aureus (S.aureus), etc.). TracrRNA may refer to a modified form of tracrRNA, which may include nucleotide changes, such as deletions, insertions or substitutions, variants, mutations or chimeras. TracrRNA can refer to a nucleic acid that is at least about 60% identical to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.) over a stretch of at least 6 consecutive nucleotides. For example, the tracrRNA sequence can be at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100% identical to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.) over a stretch of at least 6 consecutive nucleotides. Type II tracrRNA sequences can be predicted on genomic sequences by identifying regions that are complementary to partial repeat sequences in adjacent CRISPR arrays.

[0164] 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 the relevant nuclease to the target nucleic acid. The guide nucleic acid can be RNA (guide RNA or gRNA). The guide nucleic acid can be DNA. The guide nucleic acid can be a mixture of RNA and DNA. The guide nucleic acid can include crRNA or tracrRNA or a combination of the two. The guide nucleic acid can be engineered. The guide nucleic acid can be programmed to specifically bind to the target nucleic acid. A portion of the target nucleic acid can be complementary to a portion of the guide nucleic acid. The chain of the double-stranded target polynucleotide that is complementary to and hybridized with the guide nucleic acid can be referred to as a complementary chain. The chain of the double-stranded target polynucleotide that is complementary to the complementary chain and therefore may not be complementary to the guide nucleic acid can be referred to as a non-complementary chain. The guide nucleic acid can include a polynucleotide chain and can be referred to as a "single guide nucleic acid". The guide nucleic acid can include two polynucleotide chains and can be referred to as a "double guide nucleic acid". If not otherwise specified, the term "guide nucleic acid" can be included, referring to both single guide nucleic acids and double guide nucleic acids. The guide nucleic acid can include a segment that can be referred to as a "nucleic acid targeting segment" or a "nucleic acid targeting sequence" or a "spacer". The nucleic acid targeting segment may include sub-segments, which may be referred to as "protein binding segments" or "protein binding sequences" or "Cas protein binding segments."

[0165] As used herein, the terms "gene editing" and "genome editing" can be used interchangeably. Gene editing or genome editing means changing the nucleic acid sequence of a gene or genome. Genome editing can include, for example, insertions, deletions, and mutations.

[0166] In the context of two or more nucleic acid or polypeptide sequences, the term "sequence identity" or "percent identity" refers to sequences that are identical or have a specific percentage of amino acid residues or nucleotides that are identical (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) when compared and aligned for maximum correspondence within a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, for example, BLASTP using a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix with gap penalties set to 11 for presence, 1 for extension, and using a conditional composition scoring matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using a word length (W) of 2, an expected value (E) of 1000000, and a PAM30 scoring matrix (for sequences less than 30 residues, the gap penalty is set to 9 to open a gap, and to 1 to extend a gap) (these are the parameters of the BLAST suite BLASTP with default parameters, available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW with parameters; Smith-Waterman homology search algorithm with the following parameters: matches of 2, mismatches of -1, and gaps of -1; MUSCLE with default parameters; MAFFT with the following parameters: retree of 2 and maxiterations of 1000; Novafold with default parameters; HMMER hmmalign with default parameters.

[0167] The present disclosure includes variants of any enzyme in the enzymes described herein with one or more conservative amino acid substitutions. Such conservative substitutions can be carried out in the amino acid sequence of a polypeptide without destroying the three-dimensional structure or function of the polypeptide. Conservative substitutions can be completed by replacing each other with amino acids having similar hydrophobicity, polarity and R chain length. Additionally or alternatively, by comparing the alignment sequences of homologous proteins from different species, conservative substitutions can be identified by positioning the amino acid residues that mutate between species (e.g., non-conservative residues) without changing the basic function of the encoded protein. Such conservatively substituted 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 conservatively substituted variants are functional variants. Such functional variants may encompass sequences with substitutions such that the activity of key active site residues of the endonuclease is not destroyed. In some embodiments, the functional variants of any of the systems described herein lack Figure 4A , 4B and 5. In some embodiments, functional variants of any of the systems described herein lack Figure 4A , 4B and all substitutions in the conserved residues or functional residues shown in 5.

[0168] Conservative substitution tables providing functionally similar amino acids are available from various references (see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd ed. (December 1993)). The following eight groups each contain amino acids that are conservatively substituted for each other:

[0169] 1) Alanine (A), glycine (G);

[0170] 2) Aspartic acid (D), glutamic acid (E);

[0171] 3) Asparagine (N), glutamine (Q);

[0172] 4) Arginine (R), Lysine (K);

[0173] 5) Isoleucine (I), leucine (L), methionine (M), valine (V);

[0174] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0175] 7) serine (S), threonine (T); and

[0176] 8) Cysteine ​​(C), methionine (M).

[0177] As used herein, the term "RuvC_III domain" refers to the third discontinuous segment of the RuvC nuclease domain (the RuvC nuclease domain includes three discontinuous segments, RuvC_I, RuvC_II and RuvC_III). RuvC domains or their segments can usually be identified by comparing with recorded domain sequences, with the structural alignment of proteins with annotated domains, or by comparing with a Hidden Markov Model (HMM) constructed based on recorded domain sequences (e.g., Pfam HMM PF18541 of RuvC_III).

[0178] As used herein, the term "HNH domain" refers to an endonuclease domain having characteristic histidine and asparagine residues. HNH domains can generally be identified by comparison with recorded domain sequence alignments, with structural alignments of proteins with annotated domains, or by comparison with a hidden Markov model (HMM) constructed based on recorded domain sequences (e.g., Pfam HMM PF01844 for domain HNH).

[0179] As used herein, the term "recombinase" refers to an enzyme that mediates the recombination of DNA fragments located between recombinase recognition sequences, which results in excision, insertion, inversion, exchange or translocation of the DNA fragments located between the recombinase recognition sequences.

[0180] As used herein, in the context of nucleic acid modification (e.g., genomic modification), the term "recombination" refers to the process of modifying two or more nucleic acid molecules or two or more regions of a single nucleic acid molecule by the action of a recombinase protein. Recombination may particularly result in excision, insertion, inversion, exchange or translocation of nucleic acid sequences, such as in or between one or more nucleic acid molecules.

[0181] As used herein, the term "transposon" or "transposable element" refers to a nucleic acid sequence in a genome, which is a mobile genetic element that can change its location in the genome. In some cases, 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. Transposon nucleic acid sequences include, for example, genes encoding homologous transposases, one or more transposase recognition sequences, or a combination thereof. In some cases, these transposons are different in the nucleic acid type of transposition, the type of repetition at the end of the transposon, the type of cargo to be carried, or the transposition mode (i.e., self-repair or host repair) of the transposition. As used herein, the term "transposase (transposase or transposases)" refers to an enzyme that is combined with the recognition sequence of a transposon and catalyzes its movement to another part of the genome. In some cases, movement is carried out by cutting and pasting a mechanism or copying a transposition mechanism.

[0182] As used herein, the term "Tn7" or "Tn7-like transposase" refers to a family of transposases that includes three major components: a heteropolymer transposase (TnsA and / or TnsB) and a regulatory protein (TnsC). In addition to the TnsABC transposase, the Tn7 element can encode specialized target site selection proteins, TnsD and TnsE. In combination with TnsABC, the sequence-specific DNA binding protein TnsD guides transposition to a conserved site known as the "Tn7 attachment site," i.e., attTn7. TnsD is a member of a large family of proteins that also includes TniQ. TniQ has been shown to target transposition in the resolution site of a plasmid.

[0183] As used herein, the term "complex" refers to the connection of at least two components. The two components can each retain the characteristics / activity they had before forming the complex. The connection can be carried out by covalent bonding, non-covalent bonding (i.e., hydrogen bonding, ionic interactions, Van der Waals interactions, and hydrophobic bonds), using a linker, fusion, or any other suitable method. In some cases, the components in the complex are polynucleotides, polypeptides, or combinations thereof. For example, the complex can include a Cas protein and a guide nucleic acid.

[0184] In some cases, the CAST system described herein includes one or more Tn7 or Tn7-like transposases. In certain example embodiments, Tn7 or Tn7-like transposases include multimeric protein complexes. In certain example embodiments, multimeric protein complexes include TnsA, TnsB, TnsC or TniQ. In these combinations, transposases (TnsA, TnsB, TnsC, TniQ) can form complexes or fusion proteins with each other.

[0185] In some cases, the CAST system described herein includes one or more Tn5053 or Tn5053-like transposases. In certain example embodiments, Tn5053 or Tn5053-like transposases include multimeric protein complexes. In certain example embodiments, the multimeric protein complexes include TnsA, TnsB, TnsC or TniQ. In these combinations, transposases (TnsA, TnsB, TnsC, TniQ) can form complexes or fusion proteins with each other.

[0186] As used herein, the term "Cas12k" (alternatively, "class 2 VK type") refers to a subtype of a V-type CRISPR system that has been found to be defective in nuclease activity (e.g., it may include at least one defective RuvC domain that lacks at least one catalytic residue important for DNA cleavage). This effector subtype is typically associated with a CAST system.

[0187] According to IUPAC convention, the following abbreviations are used throughout the examples:

[0188] A = Adenine

[0189] C = cytosine

[0190] G = Guanine

[0191] T = thymine

[0192] R = adenine or guanine

[0193] Y = cytosine or thymine

[0194] S = guanine or cytosine

[0195] W = adenine or thymine

[0196] K = guanine or thymine

[0197] M = adenine or cytosine

[0198] B=C, G or T

[0199] D=A, G or T

[0200] H=A、C or T

[0201] V=A, C or G

[0202] Overview

[0203] The discovery of new Cas enzymes with unique functions and structures may offer the potential to further disrupt deoxyribonucleic acid (DNA) editing technologies, thereby improving speed, specificity, functionality, and ease of use. Relative to the predicted prevalence of clustered regularly interspaced 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 in part 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 may offer the potential to substantially increase the number of new CRISPR / Cas systems documented and accelerate the discovery of new oligonucleotide editing functions. A recent example of the fruitfulness of this approach was demonstrated by the discovery of the CasX / CasY CRISPR system in 2016 through metagenomic analysis of natural microbial communities.

[0204] The CRISPR / Cas system is an RNA-guided nuclease complex that has been described as acting as an adaptive immune system in microorganisms. In the natural environment of the CRISPR / Cas system, the CRISPR / Cas system appears in a CRISPR (clustered regularly spaced short palindromic repeats) operon or locus, which generally includes two parts: (i) an array of short repeats (30-40bp) separated by equally short spacer sequences, which encodes RNA-based targeting elements; and (ii) an ORF encoding Cas, which encodes a nuclease polypeptide guided by RNA-based targeting elements together with an auxiliary protein / enzyme. Efficient nuclease targeting of a specific target nucleic acid sequence generally requires the following two: (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 near the definition of the target seed (PAM is usually a sequence not often represented in the host genome). Depending on the exact function and organization of the system, CRISPR-Cas systems are generally classified into 2 categories, 5 types, and 16 subtypes based on shared functional features and evolutionary similarities (see Figure 1 ).

[0205] Class 1 CRISPR-Cas systems have large multi-subunit effector complexes and include types I, III, and IV.

[0206] Type I CRISPR-Cas systems are considered to have medium complexity in terms of components. In type I CRISPR-Cas systems, RNA targeting element arrays are transcribed into long precursor crRNAs (pre-crRNAs), which are processed at repeat elements to release short mature crRNAs, when they are followed by a suitable short consensus sequence called a protospacer adjacent motif (PAM), and the short mature crRNA guides the nuclease complex to the nucleic acid target. This treatment is carried out by an endoribonuclease subunit (Cas6) of a large endonuclease complex called a cascade, and the endonuclease complex also includes a nuclease (Cas3) protein component of the nuclease complex guided by crRNA. Cas I nucleases mainly serve as DNA nucleases.

[0207] Type III CRISPR systems may be characterized by the presence of a central nuclease called Cas10 and repeat-associated mysterious proteins (RAMPs) including Csm or Cmr protein subunits. As in type I systems, mature crRNAs are processed by pre-crRNA using a Cas6-like enzyme. Unlike type I and type II systems, type III systems appear to target and cleave DNA-RNA duplexes (such as DNA chains used as templates for RNA polymerases).

[0208] Type IV CRISPR-Cas systems have an effector complex that includes a highly reduced large subunit nuclease (csf1), two genes for the Cas5 (csf3) and Cas7 (csf2) groups of RAMP proteins, and in some cases genes for predicted small subunits; such systems are often found on endogenous plasmids.

[0209] Class 2 CRISPR-Cas systems typically have single-polypeptide multi-domain nuclease effectors and include types II, V, and VI.

[0210] Type II CRISPR-Cas systems are considered to be 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 special endonuclease subunits, but requires small trans-encoded crRNA (tracrRNA), whose region is complementary to array repeats; tracrRNA interacts with its corresponding effector nuclease (e.g., Cas9) and repeats to form a precursor dsRNA structure, which is cut by endogenous RNase III, thereby generating a mature effector enzyme loaded with both tracrRNA and crRNA. Type II nucleases are referred to as DNA nucleases. Type II effectors generally exhibit a structure consisting of a RuvC-like endonuclease domain, which is folded using RNase H, wherein an unrelated HNH nuclease domain is inserted into the fold of the RuvC-like nuclease domain. The RuvC-like domain is responsible for the cutting of the target (e.g., crRNA complementary) DNA chain, and the HNH domain is responsible for the cutting of the replaced DNA chain.

[0211] The V-type CRISPR-Cas system is characterized by a nuclease effector (e.g., Cas12) structure similar to that of the type II effector including a RuvC-like domain. Similar to type II, most (but not all) V-type CRISPR systems use tracrRNA to process pre-crRNA into mature crRNA; however, unlike the type II system that requires RNase III to cut pre-crRNA into multiple crRNAs, the V-type system is able to use the effector nuclease itself to cut pre-crRNA. Like the type II CRISPR-Cas system, the V-type CRISPR-Cas system is again referred to as a DNA nuclease. Unlike the type II CRISPR-Cas system, some V-type enzymes (e.g., Cas12a) appear to have a strong single-stranded nonspecific deoxyribonuclease activity activated by the first crRNA-directed cutting of the double-stranded target sequence.

[0212] Type VI CRISPR-Cas systems have RNA-guided RNA endonucleases. The single polypeptide effector of a type VI system (e.g., Cas13) includes two HEPN ribonuclease domains instead of a RuvC-like domain. Unlike type II and type V systems, type VI systems also do not appear to require tracrRNA to process pre-crRNA into crRNA. However, similar to type V systems, some type VI systems (e.g., C2C2) appear to have powerful single-stranded nonspecific nuclease (ribonuclease) activity activated by the first crRNA-directed cutting of the target RNA.

[0213] Class 2 CRISPR-Cas has been most widely used in engineering and development as a designer nuclease / genome editing application due to its simpler architecture.

[0214] One of the early adaptations of this system to in vitro used (i) recombinantly expressed, purified 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 desired to be cleaved, followed by a 3' tracr binding sequence (the entire crRNA was in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence); (iii) purified tracrRNA in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence; and (iv) Mg 2+ A later improved engineered system involves the crRNA of (ii) being linked to the 5' end of (iii) via a linker (e.g., GAAA) to form a single fused synthetic guide RNA (sgRNA) that can itself guide Cas9 to the target (compare Figure 2 ).

[0215] Such an engineered system can be adapted for mammalian cells by providing a DNA vector encoding: (i) an ORF encoding a codon-optimized Cas9 (e.g., a class 2 type II Cas enzyme) under a suitable mammalian promoter with 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 (having a 5' sequence starting with G followed by a 20 nt complementary targeting nucleic acid sequence linked to a 3' tracr binding sequence, a linker, and a tracrRNA sequence) under a suitable polymerase III promoter (e.g., U6 promoter).

[0216] Transposons are mobile elements that can move between locations in the genome. Such transposons have evolved to limit their negative impact on the host. A variety of regulatory mechanisms are used to maintain low-frequency transpositions, and sometimes also coordinate transpositions with various cellular processes. Some prokaryotic transposons can also mobilize functions that are beneficial to the host or otherwise help maintain the element. Some transposons may also evolve a strict control mechanism for target site selection, with the most notable example being the Tn7 family.

[0217] Transposon Tn7 and similar elements may be reservoirs for antibiotic resistance and pathogenesis functions in clinical settings, as well as encoding other adaptive functions in natural environments. For example, the Tn7 system has evolved mechanisms to almost completely avoid integration into important host genes but also to maximize dispersion of the element by recognizing mobile plasmids and phages that can move Tn7 between host bacteria.

[0218] Tn7 and Tn7-like elements can control the location and timing of their insertion, with one pathway directing insertion into a single conserved location in the bacterial genome and a second pathway that appears to be adapted to maximize targeting of mobile plasmids capable of transporting the element between bacteria (see Figure 3 The association between Tn7-like transposons and CRISPR-Cas systems suggests that transposons may hijack CRISPR effectors to generate R-loops in target loci and facilitate transposon spread via plasmids and phages.

[0219] MG64 System

[0220] In some embodiments, provided herein is a MG64 system for transposing a cargo nucleotide sequence into a target nucleic acid site. Figures 4A-4B . In some embodiments, the system comprises a double-stranded nucleic acid comprising a cargo nucleotide sequence. In some embodiments, the cargo nucleotide sequence is configured to interact with a Tn7-type or Tn5053-type transposase complex. In some embodiments, the system comprises a Cas effector complex. In some embodiments, the Cas effector complex comprises a Class 2 V-type Cas effector and an engineered guide polynucleotide configured to hybridize with a target nucleotide sequence. In some embodiments, the system comprises a Tn7-type or Tn5053-type transposase complex, the transposase complex being configured to bind to a Cas effector complex, wherein the Tn7-type or Tn5053-type transposase complex comprises a TnsB subunit.

[0221] In some cases, the cargo nucleotide sequence is flanked by a left transposase recognition sequence. In some cases, the cargo nucleotide sequence is flanked by a right transposase recognition sequence. In some cases, the cargo nucleotide sequence is flanked by a left transposase recognition sequence and a right transposase recognition sequence.

[0222] In some cases, the target nucleic acid includes a target nucleic acid site. In some cases, the target nucleic acid includes a PAM sequence that is compatible with the Cas effector complex adjacent to the target nucleic acid site. In some cases, the PAM sequence is located at 3' of the target nucleic acid site. In some cases, the PAM sequence is located at 5' of the target nucleic acid site.

[0223] In some cases, the engineered guide polynucleotide is configured to bind to the Class 2 V-type Cas effector. In some cases, the Class 2 V-type Cas effector is a Class 2 VK-type effector. In some cases, the Class 2 V-type Cas effector includes 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 to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 70% identity to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 75% identity to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 80% identity to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 85% identity to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 90% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 91% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 92% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 93% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the Class 2 type V Cas effector includes a polypeptide comprising a sequence at least about 94% identical to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220.In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 95% identity to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 96% identity to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 97% identity to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 98% identity to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector includes a polypeptide comprising a sequence having at least about 99% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector includes a polypeptide comprising a sequence having 100% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220.

[0224] In some cases, the TnsB subunit includes a polypeptide having a sequence that is 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% identical to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB subunit includes a polypeptide having a sequence that is identical to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence that is at least about 70% identical to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 75% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 80% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 85% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 90% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 91% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 92% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 93% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 94% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 95% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 96% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 97% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 98% identity to SEQ ID NOs: 2, 13, 17, and 65.In some cases, the TnsB component includes a polypeptide comprising a sequence at least about 99% identical to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence 100% identical to SEQ ID NOs: 2, 13, 17, and 65.

[0225] In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence 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% identical to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 70% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 75% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 80% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 85% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 90% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 91% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 92% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 93% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence at least about 94% identical to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111.In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 95% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 96% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 97% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 98% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence at least about 99% identical to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence 100% identical to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111.

[0226] In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, each 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 to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 70% sequence identity to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least 75% sequence identity to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 85% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 90% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 91% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111.In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 92% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 93% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 94% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 95% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 96% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 97% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 98% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 99% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include 100% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111.

[0227] In some embodiments, the systems disclosed herein include at least one engineered guide polynucleotide, such as a gRNA.

[0228] In some cases, the engineered guide polynucleotide includes a sequence comprising at least about 46-80 contiguous 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 to any one of SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 70% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 75% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 80% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 85% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 90% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 91% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least about 92% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222.In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 93% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 94% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 95% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 96% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 97% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 98% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 99% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having 100% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222.

[0229] In some cases, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 contiguous 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 to any one of SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides identical to any one of SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 70% identity to SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 75% identity to SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 80% identity to SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 85% identity to SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 90% identity to SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 contiguous nucleotides having at least about 91% identity to SEQ ID NOs:45-63, 68-75, 96-103, 123-140, and 185.In some cases, 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 to SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, 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 to SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, 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 to SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, 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 to SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, 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 to SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, 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 to SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 98% identity to SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having at least about 99% identity to SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185. In some cases, the engineered guide polynucleotide is a guide RNA and includes a sequence comprising at least about 46-80 consecutive nucleotides having 100% identity to SEQ ID NOs: 45-63, 68-75, 96-103, 123-140, and 185.

[0230] In some embodiments, the guide RNA includes various structural elements, including but not limited to: a spacer sequence, crRNA, and optional tracrRNA that binds to the original spacer sequence (target sequence). In some embodiments, the guide RNA includes a crRNA that includes a spacer sequence. In some embodiments, the guide RNA additionally includes tracrRNA or modified tracrRNA.

[0231] 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 a target sequence. For example, crRNA is or is considered to be part of a guide RNA, or is included in a guide RNA, such as a crRNA:tracrRNA chimera.

[0232] In some embodiments, the guide RNA comprises synthetic nucleotides or modified nucleotides. In some embodiments, the guide RNA comprises one or more internucleoside linkers modified by natural phosphodiester. In some embodiments, all internucleoside linkers of the guide RNA or its continuous nucleotide sequence are modified. For example, in some embodiments, the internucleoside bond comprises sulfur (S), such as a thiophosphate internucleoside bond.

[0233] In some embodiments, the guide RNA includes modifications to ribose sugar or nucleobase. In some embodiments, the guide RNA includes one or more nucleosides, the nucleosides including modified sugar moieties, wherein when compared to the ribose sugar moieties present in deoxyribonucleic acid (DNA) and RNA, the modified glucose moieties are modifications of sugar moieties. In some embodiments, the modification is within the ribose ring 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 diradical bridge between the C2 carbon and the C4 carbon on the ribose ring, or an unconnected ribose ring (e.g., UNA) that lacks a bond between the C2 carbon and the C3 carbon. In some embodiments, sugar-modified nucleosides include bicyclic hexose nucleic acids or tricyclic nucleic acids. In some embodiments, modified nucleosides include nucleosides in which the sugar moiety is replaced by a non-sugar moiety, such as a peptide nucleic acid (PNA) or a morpholino nucleic acid.

[0234] In some embodiments, the guide RNA includes one or more modified sugars. In some embodiments, sugar modifications include modifications by changing the substituents on the ribose ring to groups other than hydrogen or 2'-OH groups naturally present in DNA and RNA nucleosides. In some embodiments, the substituents are introduced at 2', 3', 4' or 5' positions or combinations thereof. In some embodiments, nucleosides with modified sugar moieties include 2' modified nucleosides, such as 2' substituted nucleosides. In some embodiments, 2' sugar-modified nucleosides are nucleosides with substituents other than -H or -OH (2' substituted nucleosides) or including 2' connected diradicals at 2' positions, and include 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 in the ribose group comprises a modification at the 2' position of the ribose group. In some embodiments, the modification at the 2' position of the ribose group is selected from the group consisting of: 2'-O-methyl, 2'-fluoro, 2'-deoxy, and 2'-O-(2-methoxyethyl).

[0235] In some embodiments, the guide RNA includes one or more modified sugars. In some embodiments, the guide RNA includes only modified sugars. In certain embodiments, the guide RNA includes greater 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 includes 2'-O-methoxyethyl. In some embodiments, the guide RNA includes both internucleoside linker modifications and nucleoside modifications.

[0236] In some cases, the guide RNA includes a sequence complementary to a eukaryotic, fungal, plant, mammalian or human genomic polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a fungal genomic polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a plant genomic polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a mammalian genomic polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a human genomic polynucleotide sequence.

[0237] 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 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, 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 In the present invention, the present invention relates to a polypeptide having at least one nucleotide sequence and at least one nucleotide sequence. In the present invention, the present invention relates to a polypeptide having at least one nucleotide sequence and at least one nucleotide sequence. In the present invention, the present invention relates to a polypeptide having at least one nucleotide sequence and at least one nucleotide sequence.

[0238] In some cases, the left recombinase sequence comprises 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 to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having at least about 70% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having at least about 75% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having at least about 80% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having at least about 85% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having at least about 90% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having at least about 91% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having at least about 92% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence includes a sequence having at least about 93% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence includes a sequence having at least about 94% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence includes a sequence having at least about 95% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence includes a sequence having at least about 96% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence includes a sequence having at least about 97% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence includes a sequence having at least about 98% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78.In some cases, the left recombinase sequence comprises a sequence having at least about 99% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having 100% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78.

[0239] In some cases, the right recombinase sequence comprises 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 to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 70% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 75% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 80% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 85% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 90% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 91% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 92% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 93% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 94% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 95% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 96% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence includes a sequence that is at least about 97% identical to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93.In some cases, the right recombinase sequence comprises a sequence having at least about 98% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 99% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having 100% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93.

[0240] In some cases, the class 2 type V Cas effector and Tn7-type transposase complex is 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.

[0241] In some embodiments, the Class 2 Type V effector comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is located at the N-terminus of the Class 2 Type V effector. In some embodiments, the NLS is located at the C-terminus of the Class 2 Type V effector. In some embodiments, the NLS is located at the N-terminus and the C-terminus of the Class 2 Type V effector.

[0242] In some embodiments, the NLS comprises a sequence of any one of SEQ ID NOs: 192-207, 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 to any one of SEQ ID NOs: 192-207. In some cases, the NLS comprises a sequence having at least about 80% identity to SEQ ID NOs: 192-207. In some cases, the NLS comprises a sequence having at least about 85% identity to SEQ ID NOs: 192-207. In some cases, the NLS comprises a sequence having at least about 90% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence having at least about 91% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence having at least about 92% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence having at least about 93% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence having at least about 94% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence having at least about 95% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence having at least about 96% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence having at least about 97% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence that is at least about 98% identical to SEQ ID NOs: 192-207. In some cases, the NLS comprises a sequence that is at least about 99% identical to SEQ ID NOs: 192-207. In some cases, the NLS comprises a sequence that is 100% identical to SEQ ID NOs: 192-207.

[0243] Table 1: Exemplary NLS sequences

[0244]

[0245]

[0246] In some embodiments, the Cas effector complex further includes a 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 to any one of SEQ ID NOs: 181-183. In some cases, S15 is encoded by a sequence having at least about 70% identity to SEQ ID NOs: 181-183. In some cases, S15 is encoded by a sequence having at least about 75% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 80% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 85% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 90% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 91% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 92% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 93% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 94% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 95% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 96% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 97% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 98% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 99% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having 100% identity to SEQ ID NO: 181-183.

[0247] In some cases, S15 includes a sequence with at least about 70% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 75% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 80% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 85% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 90% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 91% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 92% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 93% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 94% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 95% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 96% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 97% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 98% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 99% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with 100% identity to SEQ ID NO: 187-189.

[0248] In some embodiments, the Cas effector complex comprises one or more linkers connecting type 2 V-type effectors, small prokaryotic ribosomal protein subunit S15, transposase, gRNA, or a combination thereof. In some embodiments, the linker comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, or 400 amino acids. In some embodiments, the linker comprises 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 linker is encoded by the sequence of SEQ ID NO: 186, or a sequence that is 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% identical to SEQ ID NO: 186. In some embodiments, the linker is encoded by SEQ ID NO: 186.

[0249] Fusion Protein

[0250] In some embodiments, described herein is 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 the fusion protein comprises a 2-type V-type effector, a small prokaryotic ribosomal protein subunit S15, a transposase, a gRNA or a combination thereof. In some embodiments, the fusion protein comprises one or more transposases.

[0251] In some embodiments, a nuclear localization sequence (NLS) is fused to the Class 2 Type V effector. In some embodiments, the NLS is fused at the N-terminus of the Class 2 Type V effector. In some embodiments, the NLS is fused at the C-terminus of the Class 2 Type V effector. In some embodiments, the NLS is fused at both the N-terminus and the C-terminus of the Class 2 Type V effector.

[0252] In some embodiments, the NLS comprises a sequence of any one of SEQ ID NOs: 192-207, 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 to any one of SEQ ID NOs: 192-207. In some cases, the NLS comprises a sequence having at least about 80% identity to SEQ ID NOs: 192-207. In some cases, the NLS comprises a sequence having at least about 85% identity to SEQ ID NOs: 192-207. In some cases, the NLS comprises a sequence having at least about 90% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence having at least about 91% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence having at least about 92% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence having at least about 93% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence having at least about 94% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence having at least about 95% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence having at least about 96% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence having at least about 97% identity to SEQ ID NO: 192-207. In some cases, the NLS comprises a sequence that is at least about 98% identical to SEQ ID NOs: 192-207. In some cases, the NLS comprises a sequence that is at least about 99% identical to SEQ ID NOs: 192-207. In some cases, the NLS comprises a sequence that is 100% identical to SEQ ID NOs: 192-207.

[0253] In some embodiments, the fusion protein or nucleic acid encoding the fusion protein includes 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 the N-terminus and C-terminus of S15.

[0254] In some embodiments, the S15 fusion protein further comprises a cleavable peptide. In some embodiments, the peptide is a 2A peptide.

[0255] In some embodiments, the S15 fusion protein is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 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 to any one of SEQ ID NOs: 181-183. In some embodiments, the Cas effector complex further includes a small prokaryotic ribosomal protein subunit S15. In some embodiments, the S15 fusion protein is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 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 to any one of SEQ ID NOs: 181-183. In some cases, S15 is encoded by a sequence having at least about 70% identity to SEQ ID NOs: 181-183. In some cases, S15 is encoded by a sequence having at least about 75% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 80% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 85% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 90% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 91% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 92% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence having at least about 93% identity to SEQ ID NO: 181-183. In some cases, S15 is encoded by a sequence that is at least about 94% identical to SEQ ID NOs: 181-183.In some cases, S15 is encoded by a sequence having at least about 95% identity to SEQ ID NOs: 181-183. In some cases, S15 is encoded by a sequence having at least about 96% identity to SEQ ID NOs: 181-183. In some cases, S15 is encoded by a sequence having at least about 97% identity to SEQ ID NOs: 181-183. In some cases, S15 is encoded by a sequence having at least about 98% identity to SEQ ID NOs: 181-183. In some cases, S15 is encoded by a sequence having at least about 99% identity to SEQ ID NOs: 181-183. In some cases, S15 is encoded by a sequence having 100% identity to SEQ ID NOs: 181-183.

[0256] In some embodiments, the S15 fusion protein comprises a sequence having at least about 70% sequence identity to any one of SEQ ID NOs: 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 to any one of SEQ ID NOs: 187-189. In some cases, S15 comprises a sequence having at least about 70% identity to SEQ ID NOs: 187-189. In some cases, S15 includes a sequence with at least about 75% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 80% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 85% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 90% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 91% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 92% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 93% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 94% identity to SEQ ID NO: 187-189. In some cases, S15 includes a sequence with at least about 95% identity to SEQ ID NOs: 187-189. In some cases, S15 includes a sequence with at least about 96% identity to SEQ ID NOs: 187-189. In some cases, S15 includes a sequence with at least about 97% identity to SEQ ID NOs: 187-189. In some cases, S15 includes a sequence with at least about 98% identity to SEQ ID NOs: 187-189. In some cases, S15 includes a sequence with at least about 99% identity to SEQ ID NOs: 187-189. In some cases, S15 includes a sequence with 100% identity to SEQ ID NOs: 187-189.

[0257] In some embodiments, the NLS is fused to 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 the N-terminus and the C-terminus of the transposase.

[0258] In some embodiments, the fusion protein or nucleic acid encoding the fusion protein includes a gRNA described herein (e.g., a dual gRNA or a single gRNA).

[0259] In some embodiments, the class 2 V-type effector, small prokaryotic ribosomal protein subunit S15, transposase, gRNA or fusion protein comprises a tag. In some embodiments, the tag is an affinity tag. In some embodiments, the tag is a polypeptide or a polynucleotide. Exemplary affinity tags include but are not limited to His tags, Flag tags, Myc tags, MBP tags and GST tags.

[0260] In some embodiments, the class 2 type V effector, small prokaryotic ribosomal protein subunit S15, transposase or fusion protein comprises a protease cleavage site. Exemplary protease cleavage sites include but are not limited to TEV sites, C3 sites, factor Xa sites and enterokinase sites.

[0261] cell

[0262] In certain embodiments, described herein is a cell comprising a system described herein.

[0263] In some embodiments, the cell is a eukaryotic cell (e.g., a plant cell, an animal cell, a protist cell, or a fungal cell); a mammalian cell (a Chinese hamster ovary (CHO) cell, a baby hamster kidney (BHK), a human embryonic kidney (HEK), a mouse myeloma (NS0), or a human retinal cell); an immortalized cell (e.g., a HeLa cell, a COS cell, a HEK-293T cell, a MDCK cell, a 3T3 cell, a PC12 cell, a Huh7 cell, a HepG2 cell, a K562 cell, a N2a cell, or a SY5Y cell); an insect cell (e.g., a Spodoptera frugiperda cell, a Trichoplusia ni cell, a Drosophila melanogaster cell, a S2 cell, or a Heliothis virescens cell); a yeast cell (e.g., a Saccharomyces cerevisiae cell, a cerevisiae) cells, Cryptococcus cells, or Candida cells); plant cells (e.g., thin-walled 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 cell is a eukaryotic cell. 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.

[0264] 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, a primary cell, or a derivative thereof.

[0265] Delivery and vectors

[0266] In some embodiments, disclosed herein is a nucleic acid sequence encoding an MG64 system comprising a class 2 V-type effector, a small prokaryotic ribosomal protein subunit S15, a transposase, a gRNA, a fusion protein, or a gene editing system disclosed herein.

[0267] In some embodiments, the nucleic acid encoding the MG64 system is DNA, such as linear DNA, plasmid DNA, or minicircle DNA. In some embodiments, the nucleic acid encoding the MG64 system is RNA, such as mRNA.

[0268] 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 replicate autonomously in a cell), a cosmid (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 phagemid, a phage derivative, a bacmid, or a virus. In some embodiments, the nucleic acid-based vector is selected from the list consisting of: 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.

[0269] In some embodiments, the nucleic acid-based vector includes a promoter. In some embodiments, the promoter is selected from the group consisting of: a mini promoter, an inducible promoter, a constitutive promoter, 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 a U6 promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter is encoded by the sequence of any one of SEQ ID NOs: 190-191, or a sequence that is 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% identical to any one of SEQ ID NOs: 190-191.

[0270] In some embodiments, the nucleic acid-based vector is a virus. In some embodiments, the virus is an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a dengue virus, a lentivirus, a herpes virus, a poxvirus, anellovirus, a bocavirus, a vaccinia virus, or a retrovirus. In some embodiments, the virus is an alphavirus. In some embodiments, the virus is a parvovirus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is an AAV. In some embodiments, the virus is a baculovirus. In some embodiments, the virus is a dengue virus. In some embodiments, the virus is a lentivirus. In some embodiments, the virus is a herpes virus. In some embodiments, the virus is a poxvirus. 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.

[0271] 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 herpes virus is HSV type 1, HSV type 2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.

[0272] 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.

[0273] 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.

[0274] In some embodiments, the nucleic acid encoding the MG64 system is delivered by a delivery system (e.g., a non-viral delivery system) based on non-nucleic acids. In some embodiments, the non-viral delivery system is a liposome. In some embodiments, the nucleic acid is associated with a lipid. In some embodiments, the nucleic acid associated with a lipid can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, connected to the liposome via a connecting molecule associated with the liposome and the nucleic acid, captured in the liposome, compounded with the liposome, dispersed in a solution containing lipids, mixed with lipids, merged with lipids, contained as a suspension in the lipid, containing micelles or compounded with micelles, or otherwise associated with lipids. In some embodiments, the nucleic acid is included in a lipid nanoparticle (LNP).

[0275] In some embodiments, the fusion protein or genome editing system is stably or transiently introduced into the cell in any suitable manner. In some embodiments, the fusion protein or genome editing system is transfected into the cell. In some embodiments, the cell is transduced or transfected with a nucleic acid construct encoding a fusion protein or genome editing system. For example, the cell is transduced (for example, with a virus encoding a fusion protein or genome editing system), or the fusion protein or genome editing system (for example, with a plasmid encoding a fusion protein or genome editing system) is transfected with a nucleic acid encoding a fusion protein or genome editing system or a translated fusion protein or genome editing system. In some embodiments, transduction is a stable or transient transduction. In some embodiments, for example, when the fusion protein or genome editing system includes a CRISPR nuclease, the cell expressing the fusion protein or genome editing system or containing the fusion protein or genome editing system is transduced or transfected with one or more gRNA molecules. In some embodiments, the plasmid expressing the fusion protein or genome editing system is introduced into the cell by electroporation, transient (for example, lipid infection) and stable genome integration (for example, piggybac) and viral transduction (for example, slow virus or AAV) or other methods known to those skilled in the art. In some embodiments, the gene editing system is introduced into the cell as one or more polypeptides. In some embodiments, delivery is achieved by using RNP complexes. Methods for delivery of polypeptides and / or RNPs to cells are known in the art, such as by electroporation or by cell squeezing.

[0276] Exemplary delivery methods for nucleic acids include lipofection, nucleofection, electroporation, stable genomic integration (e.g., piggybac), microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycationic or lipid nucleic acid conjugates, naked DNA, artificial virus particles, and agent-enhanced DNA uptake. Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam TM 、Lipofectin TM and SF cell line 4D-Nucleofectin X Kit TM (Lonza). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include cationic lipids and neutral lipids in WO 91 / 17424 and WO 91 / 16024. In some embodiments, delivery to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). In some embodiments, the nucleic acid is included in a liposome or nanoparticle that specifically targets a host cell.

[0277] Additional methods of delivering nucleic acids to cells are known to those skilled in the art. See, for example, US 2003 / 0087817.

[0278] In some embodiments, the disclosure provides a cell comprising a 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.

[0279] Methods for transposition

[0280] The present disclosure provides methods for transposing a cargo nucleotide sequence into a target nucleic acid site. In some embodiments, the method comprises expressing the system described herein in a cell or introducing the system described herein into a cell. In some embodiments, the method comprises contacting a cell with the system described herein.

[0281] In some embodiments, the method comprises contacting a double-stranded nucleic acid comprising a cargo nucleotide sequence with a Cas effector complex, the Cas effector complex comprising a Class 2 V-type Cas effector and at least one engineered guide polynucleotide configured to hybridize with a target nucleotide sequence. In some embodiments, the method comprises contacting a double-stranded nucleic acid comprising a cargo nucleotide sequence with 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 method comprises contacting a double-stranded nucleic acid comprising a cargo nucleotide sequence with a double-stranded target nucleic acid comprising a target nucleic acid site.

[0282] In some cases, the cargo nucleotide sequence is flanked by a left transposase recognition sequence. In some cases, the cargo nucleotide sequence is flanked by a right transposase recognition sequence. In some cases, the cargo nucleotide sequence is flanked by a left transposase recognition sequence and a right transposase recognition sequence. In some cases, the method further comprises a PAM sequence compatible with the Cas effector complex adjacent to the target nucleic acid site. In some cases, the PAM sequence is located at 3' of the target nucleic acid site.

[0283] In some cases, the engineered guide polynucleotide is configured to bind to the Class 2 type V Cas effector. In some cases, the Class 2 type V Cas effector includes 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 to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 70% identity to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 75% identity to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 80% identity to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 85% identity to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 90% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 91% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 92% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 93% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the Class 2 type V Cas effector includes a polypeptide comprising a sequence at least about 94% identical to SEQ ID NO: 1, 12, 16, 20-30, 64, 80-85, and 220.In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 95% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 96% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 97% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 98% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector includes a polypeptide comprising a sequence having at least about 99% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220. In some cases, the class 2 type V Cas effector includes a polypeptide comprising a sequence having 100% identity to SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220.

[0284] In some cases, the TnsB subunit includes a polypeptide having a sequence that is 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% identical to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsA subunit includes a polypeptide having a sequence that is identical to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence that is at least about 70% identical to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 75% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 80% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 85% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 90% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 91% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 92% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 93% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 94% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 95% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 96% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 97% identity to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence having at least about 98% identity to SEQ ID NOs: 2, 13, 17, and 65.In some cases, the TnsB component includes a polypeptide comprising a sequence at least about 99% identical to SEQ ID NOs: 2, 13, 17, and 65. In some cases, the TnsB component includes a polypeptide comprising a sequence 100% identical to SEQ ID NOs: 2, 13, 17, and 65.

[0285] In some cases, the Tn7-type transposase complex includes at least one polypeptide (e.g., at least 1, 2, 3, 4, 5, 6, or more than 6 polypeptides) comprising a sequence 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% identical to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 70% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 75% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 80% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 85% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 90% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 91% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 92% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 93% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence at least about 94% identical to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111.In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 95% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 96% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 97% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence having at least about 98% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence at least about 99% identical to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes a polypeptide comprising a sequence 100% identical to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111.

[0286] In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, each independently comprising a polypeptide having a sequence 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% identical to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least 75% sequence identity to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some embodiments, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 85% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 90% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 91% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111.In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 92% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 93% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 94% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 95% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 96% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 97% identity to SEQ ID NO: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 98% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include at least about 99% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently include 100% identity to SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111.

[0287] In some cases, the engineered guide polynucleotide includes a sequence comprising at least about 46-80 contiguous 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 to any one of SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 70% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 75% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 80% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 85% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 90% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 91% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least about 92% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222.In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 93% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 94% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 95% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 96% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 97% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 98% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 99% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having 100% identity to SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222.

[0288] In some cases, the left recombinase sequence comprises 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 to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having at least about 70% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having at least about 75% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having at least about 80% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having at least about 85% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having at least about 90% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having at least about 91% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having at least about 92% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence includes a sequence having at least about 93% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence includes a sequence having at least about 94% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence includes a sequence having at least about 95% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence includes a sequence having at least about 96% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence includes a sequence having at least about 97% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence includes a sequence having at least about 98% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78.In some cases, the left recombinase sequence comprises a sequence having at least about 99% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78. In some cases, the left recombinase sequence comprises a sequence having 100% identity to SEQ ID NOs: 9, 11, 36-38, 76, and 78.

[0289] In some cases, the right recombinase sequence comprises 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 to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 70% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 75% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 80% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 85% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 90% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 91% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 92% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 93% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 94% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 95% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 96% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence includes a sequence that is at least about 97% identical to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93.In some cases, the right recombinase sequence comprises a sequence having at least about 98% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having at least about 99% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93. In some cases, the right recombinase sequence comprises a sequence having 100% identity to SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93.

[0290] In some cases, the class 2 type V Cas effector and Tn7-type transposase complex is 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.

[0291] use

[0292] The systems disclosed herein can be used for various 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 a subject's disease; inactivate a gene to determine its function in a cell; as a diagnostic tool for detecting pathogenic genetic elements (e.g., by cleaving retroviral RNA or amplified DNA sequences encoding pathogenic mutations); as an inactivating enzyme combined with a probe to target and detect specific nucleotide sequences (e.g., sequences encoding bacterial antibiotic resistance); inactivate a virus or make it unable to infect a host cell by targeting the viral genome; add genes or modify metabolic pathways to engineer an organism to produce valuable small molecules, macromolecules, or secondary metabolites; establish gene drive elements for evolutionary selection and / or as a biosensor to detect interference of foreign small molecules and nucleotides with cells.

[0293] Reagent test kit

[0294] In some embodiments, the present 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 a nucleotide sequence encoding a fusion protein or a component of a genome editing system capable of modifying a target DNA sequence. In some embodiments, the nucleotide sequence includes a heterologous promoter driving the expression of an RNA genome editing system component.

[0295] In some embodiments, a class 2 type V effector, a small prokaryotic ribosomal protein subunit S15, a transposase, a gRNA or a fusion protein, or a gene editing system comprising any combination thereof disclosed herein is assembled into a pharmaceutical, diagnostic or research kit to facilitate its use in therapeutic, diagnostic or research applications. The kit may include one or more containers containing any vector disclosed herein and instructions for use.

[0296] Kit can be designed to help researchers use the methods described herein, and can take many forms. Each composition of the kit, where applicable, can be provided in liquid form (e.g., in solution form) or in solid form (e.g., dry powder). In some cases, some compositions can be configurable 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), the composition can be provided together with the kit, or it may not be provided together with the kit. As used herein, "instructions" can define the composition of instructions and / or promotion, and are generally related to written instructions on or associated with the packaging of the present disclosure. Instructions can also include any oral or electronic instructions provided in any way, so that the user will clearly recognize that the instructions will be related to the kit, for example, audio-visual (e.g., videotapes, DVDs, etc.), the Internet and / or network-based communications, etc. In certain embodiments, the written instructions are in the form of regulations of government agencies regulating the manufacture, use or sale of drugs or biological products, and the instructions can also reflect the permission of the agency for animal administration in terms of manufacture, use or sale.

[0297] Examples

[0298] The following examples are given for the purpose of showing various embodiments of the present disclosure and are not meant to limit the present disclosure in any way. The present examples and the methods described herein currently represent preferred embodiments, are exemplary, and are not intended to limit the scope of the present disclosure. Those skilled in the art will appreciate changes therein and other uses encompassed within the spirit of the present disclosure as defined by the scope of the claims.

[0299] Example 1 - (General Protocol) PAM sequence identification / confirmation of the system described herein

[0300] Putative endonucleases are expressed in an expression system based on E. coli lysate. The PAM sequence is determined by sequencing a plasmid containing a randomly generated potential PAM sequence that can be cut by the putative nuclease. In this system, an E. coli codon-optimized nucleotide sequence encoding a putative nuclease is transcribed and translated in vitro from a PCR fragment under the control of a T7 promoter. A second PCR fragment with a minimal CRISPR array consisting of a T7 promoter and a subsequent repeat-spacer-repeat sequence is transcribed in the same reaction. Successful expression of the endonuclease and repeat-spacer-repeat sequence in an expression system based on E. coli lysate, followed by CRISPR array treatment, provides an active in vitro CRISPR nuclease complex.

[0301] The library of the target plasmid containing the spacer sequence is incubated with the expression system reaction output, and the spacer sequence matches the spacer sequence before the 8N mixed base (potential PAM sequence) in the minimum array. After 1-3 hours, the reaction is stopped, and DNA is recovered by a DNA cleaning kit. The adapter sequence is flat-ended and connected to the DNA with the active PAM sequence cut by an endonuclease, and the uncut DNA cannot be close to the connection. Then the DNA segment including the active PAM sequence is amplified by PCR with primers specific to the library and the adapter sequence. The PCR amplification product is parsed on a gel to identify the amplicon corresponding to the cutting event. The amplification segment of the cutting reaction is also used as a template for preparing an NGS library or as a substrate for Sanger sequencing. This resulting library is sequenced, and the resulting library is a subset of the starting 8N library, revealing a sequence with a PAM activity compatible with the CRISPR complex. For PAM testing with treated RNA constructs, the same procedure was repeated except that in vitro transcribed RNA was added along with the plasmid library and the minimal CRISPR array template was omitted.

[0302] Analysis of the intergenic regions surrounding the Cas effectors and CRISPR array identified potential anti-repeat sequences corresponding to the tracrRNA duplex sequence. The tracrRNA and crRNA repeat sequences were folded and trimmed, and a GAAA tetraloop sequence was added to maintain the stem-loop region of the crRNA-tracrRNA complex.

[0303] Example 2A - Targeting Integrase Activity In Vitro

[0304] Integrase activity is determined using previously identified PAMs, but can alternatively be performed with PAM library substrates, where efficiency is reduced. One arrangement of components for in vitro testing involves three plasmids in addition to the donor sequence: (1) an expression plasmid with an effector (or multiple effectors) under a T7 promoter; (2) an expression plasmid with a transposase gene under a 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 end (LE) and right end (RE) DNA sequences for transposition around a cargo gene (e.g., a selection marker, such as a Tet resistance gene). Using an in vitro transcription / translation system (e.g., a system based on E. coli lysate or reticulocyte lysate), the effector and transposase genes are expressed. After expression, RNA, target DNA, and donor DNA are added and incubated to allow transposition to occur. Transposition was detected by PCR across the junction of the transposase site, 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 targeting site. The primers are located downstream to accommodate and detect various insertion sites. Primers are designed to detect integration in either orientation of the cargo and on either side of the spacer, as the integration direction was not previously recorded.

[0305] Integration efficiency was measured by quantitative PCR (qPCR) measuring the experimental output of target DNA and integrated cargo, which was normalized to the amount of unmodified target DNA also measured by qPCR.

[0306] This assay can be performed with purified protein components rather than from lysate-based expression. In this case, proteins were expressed in E. coli protease-deficient B strains under a T7 inducible promoter, cells were lysed using sonication, and the His-tagged proteins of interest were 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, 5% glycerol; pH 7.5 (or other buffers as determined for maximum stability) and stored at -80 °C. After purification, the effector and transposase are added to a reaction buffer containing sgRNA, target DNA, and donor DNA as described above, e.g., 26 mM HEPES pH 7.5, 4.2 mM TRIS pH 8, 50 μg / mL BSA, 2 mM ATP, 2.1 mM DTT, 0.05 mM EDTA, 0.2 mM MgCl2, 28 mM NaCl, 21 mM KCl, 1.35% glycerol (final pH 7.5) supplemented with 15 mM Mg(Oac)2.

[0307] Example 2B - In vitro activity

[0308] Targeted nuclease

[0309] In situ expression and protein sequence analysis indicated that some RNA-guided effectors are active nucleases. They contain predicted endonuclease-related domains (matching RuvC and HNH_endonuclease domains) and / or predicted HNH and RuvC catalytic residues.

[0310] Using an E. coli lysate-based expression system and in vitro transcribed RNA, candidate activities were tested with engineered single guide RNA sequences. Active proteins that successfully cleaved the library produced a band of approximately 170 bp in the gel.

[0311] DNA integration and transposition

[0312] When the genomic sequence of the encoding transposon contains one or more protein sequences with transposase and / or integrase function in the left and right ends of the transposon, it is predicted that the transposon is active. As defined herein, the Tn7 transposon can include catalytic transposase TnsB, but can also contain TnsA, TnsC, TnsD, TnsE, TniQ and / or other transposases or integrase. The transposon end includes a predicted transposase binding site, which contains direct and / or inverted repeats of 15bp to 150bp in length, flanking transposase protein and other "goods" genes. Protein sequence analysis indicates that the transposase contains integrase domain, transposase domain and / or transposase catalytic residues, showing that they are active (for example, Figure 4A ).

[0313] Targeted DNA integration

[0314] Putative CRISPR-associated transposons (CASTs) contain DNA and / or RNA that targets CRISPR nucleases or effectors, as well as proteins with predicted transposase function in the vicinity of the CRISPR array. In some systems, nucleases are predicted to be active based on the presence of endonuclease-associated catalytic domains and / or catalytic residues.

[0315] In some systems, the predicted effector has homology to a documented CRISPR effector protein but is inactive based on the absence of the endonuclease domain and / or catalytic residues. When the CRISPR locus (inactive CRISPR nuclease and array) and the transposase protein are located within the left and right ends of the predicted transposon, the predicted transposase is associated with the effector ( Figure 4A ). In this case, effectors are predicted to direct DNA integration to a specific genomic location based on the guide RNA.

[0316] CAST activity was tested with 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 the PAM corresponding to the Cas enzyme; (3) donor DNA fragments containing DNA markers or fragments flanked by LE and RE of the transposase system in the DNA fragment or plasmid; (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 the donor fragment that successfully transposed was determined by PCR amplification of the donor-target junction.

[0317] After the transposition reaction was performed, PCR amplification of the junction showed that proper donor-target formation had occurred and that the transposition reaction was sg-dependent. Figure 6). PCR amplifications for reactions #3 and #4 indicated two orientations of the donor relative to the target: one in which the LE was closer to the PAM, and one in which the RE was closer to the PAM. Although both transposition orientations were performed, there was a preference for donor integration in the target in which the LE was closer to the PAM, as indicated by the strong bands present for reactions #4 and #5.

[0318] Sanger sequencing of the preferred oriented products was performed. In integrations that occurred with the LE closer to the PAM, the sequencing chromatogram signal was clearly attenuated from either the forward or reverse orientation at the target / donor junction. This suggests that in products oriented with the LE closer to the PAM, integration occurred over a range of nucleotides, with the major product of the LE product closer to the PAM integrated as 61 bp from the PAM ( Fig. 7A Donor-derived sequencing at the donor-target junction defines the composition of the basic universe of LE and RE sequences ( Figure 7A-7B Further investigation of the LE and RE domains may determine the internal limits of the LE and RE sequences that are critical for transposition. Sequencing of the RE on the LE product closer to the PAM showed a 3 bp duplication downstream of the donor RE ( Figure 7B ). This is due in part to Tn7 transposase integration events that cleave and ligate the donor fragment at staggered cleavage sites. The 3 bp repeat is smaller than the expected 5 bp repeat from other Tn7 transposases.

[0319] Sanger sequencing of PCR amplification products on the 8N library of the target plasmid also elucidated the PAM preference of the MG64-1 effector as nGTn / nGTt on the 5' end of the spacer ( Figure 7C ). NGS analysis of PAM library targets confirmed the specificity of the nGTn motif at the 5' end.

[0320] Example 3 - Predicting RNA folding

[0321] The predicted RNA folds of active single RNA sequences were calculated at 37°C using the method of Andronescu 2007. All hairpin loop secondary structures were individually deleted from the structure and iteratively compiled into smaller single guides. In the second approach, the tracrRNA of MG64-1 was aligned with the recorded Vk-type tracrRNA, and the uniquely inserted regions were mutated out of the single guide and reduced to a maximum of 57 bases. Fig. 12A The predicted structure of the MG64-1 sgRNA is depicted. Fig. 12B The predicted structure of MG64-3 sgRNA is depicted. Fig. 12C The predicted structure of the MG64-5 sgRNA is depicted. The color of the base corresponds to the probability of base pairing of the base, where red indicates high probability and blue indicates low probability.

[0322] Example 4 - Transposon end verification by gel shift

[0323] The TnsB binding of the transposon end is tested by electrophoretic mobility shift assay (EMSA). In this case, potential LE or RE is synthesized into DNA fragments (100-500bp), and end-labeled with FAM by PCR with primers marked by FAM. TnsB protein is synthesized in in vitro transcription / translation system. After synthesis, 1 μL TnsB protein is added to 50nM RE or LE in 10 μL reactions in binding buffer (20mM HEPES pH 7.5, 2.5mM Tris pH7.5, 10mM NaCl, 0.0625mM EDTA, 5mM TCEP, 0.005% BSA, 1ug / mL poly (dI-dC) and 5% glycerol). Will be combined at 30 ° of incubations for 40 minutes, then add 2uL 6X loading buffer (60mM KCl, 10mM Tris pH 7,6, 50% glycerol). Binding reactions were separated and visualized on 5% TBE gels. Shifts in LE or RE in the presence of TnsB were attributed to successful binding and indicated transposase activity ( Fig.24 ).

[0324] Example 5 - Integrase activity in E. coli

[0325] Since E. coli lacks the ability to effectively repair double-strand DNA breaks in the genome, transformation of E. coli with agents that can cause double-strand breaks in the E. coli genome results in cell death. Taking advantage of this phenomenon, endonuclease or effector-assisted integrase activity is tested in E. coli by recombinantly expressing the endonuclease or effector-assisted integrase and guide RNA (e.g., as determined in Example 3) in the target strain, wherein the spacer / target and PAM sequences are integrated into its genomic DNA.

[0326] The engineered strains are then transformed with plasmids containing nucleases or effectors with single guide RNAs, plasmids expressing integrase and helper genes, and plasmids containing temperature-sensitive replication origins with selectable markers flanking left (LE) and right (RE) transposon motifs for integration. Transformants that induce expression of these genes are then screened by selecting for plasmid replication at the restrictive temperature to transfer the markers to the genomic target, and marker integration in the genome is confirmed by PCR.

[0327] An unbiased approach was used to screen for off-target integration. In short, purified gDNA was fragmented with Tn5 transposase or shearing, and the DNA of interest was PCR amplified using primers specific for the attached adapter and selectable marker. Amplicons were then prepared for NGS sequencing. Analysis of the resulting sequences trimmed the transposon sequence, and the flanking sequences were mapped to the genome to determine the insertion position, and the off-target insertion rate was determined.

[0328] Example 6-Colony PCR Screening for Transposase Activity

[0329] To test nuclease or effector-assisted integrase activity in bacterial cells, strain MGB0032 was constructed from BL21 (DE3) E. coli cells engineered to contain a target specific for MG64_1 and the corresponding PAM sequence. MGB0032 E. coli cells were then transformed with pJL56 (plasmid expressing MG64_1 effector and auxiliary kit, ampicillin resistance) and pTCM 64_1sg (chloramphenicol resistance plasmid expressing a single guide RNA sequence of the engineered target of interest driven by a T7 promoter).

[0330] Then the MGB0032 culture containing two plasmids is grown to saturation, diluted at least 1:10 into the growth culture with appropriate antibiotics, and incubated at 37°C until OD is about 1. Make the cells from this growth stage have electrocompetence and transform with streamlined 64_1p donor, which is a plasmid with a tetracycline resistance marker flanking the left end (LE) and right end (RE) transposon motif for integration. Then in the presence or absence of IPTG with a final concentration of 100 μM, the electroporated cells are recovered on LB medium for 2 hours, then plated on LB-agar-ampicillin-chloramphenicol-tetracycline and incubated at 37°C for 4 days. Use a sterile toothpick to sample each resulting CFU mixed into water. To this solution was added Q5 High Fidelity PCR Master Mix (New England Biolabs) and primers LA155 (5'-GCTCTTCCGATCTNNNNNGATGAGCGCATTGTTAGATTTCAT-3') and oJL50 (5'-AAACCGACATCGCAGGCTTC-3'). These primers flank the predicted insertion junction. The predicted product size is 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.

[0331] Example 7 - Intracellular expression / in vitro assay

[0332] To test the function of the NLS construct in a physiologically relevant environment, constructs cloned with active NLS-tagged CAST components were integrated into K562 cells using lentiviral transduction. In short, the constructs cloned into the lentiviral transfer plasmid were transfected into 293T cells with encapsulation and packaging plasmids, and the virus containing the supernatant was collected from the culture medium after 72 hours of incubation. The culture medium containing the virus was then incubated with the K562 cell line and 8 μg / mL polybrene for 72 hours, and then the transfected cells were selected and batch integration was performed using 1 μg / mL of puromycin for 4 days. The selected cell lines were collected at the end of 4 days, and differential lysis was performed for nuclear and cytoplasmic fractions. The transposition ability of subsequent fractions and a set of complementary in vitro expression components was then tested.

[0333] 10 million cells were centrifuged and washed once with 1x PBS pH 7.4. The supernatant wash was completely aspirated into the cell pellet and quickly frozen at -80 ° C for 16 hours. After thawing on ice, the cell pellet size was measured by mass, and the cell fraction and nuclear extraction reagent (NE-PER) of the appropriate extraction volume were used to naturally extract the protein in the cell fraction. In short, the cytoplasmic extraction reagent was used with a cell mass of 1:10 and the extraction reagent volume. The cell suspension was mixed by vortexing and lysed with a non-ionic detergent. The cells were then centrifuged at 16,000xg 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 by the original cell mass of 1:2, and incubated on ice for 1 hour by intermittent vortexing on ice. The nuclear suspension was then centrifuged at 16,000xg for 10 minutes at 4 ° C, and the supernatant nuclear extract was decanted and tested for in vitro transposition activity. For each condition, 4 μL of each cell and nuclear extract was used for in vitro transposition reactions with a complementary set of in vitro expressed proteins, donor DNA, ptarget, and buffer. Evidence of transposition activity was determined by PCR amplification of the donor-target junction.

[0334] Example 8 - Activity in mammalian cells (predictive)

[0335] In order to show the targeting and cutting activity in mammalian cells, the nuclear localization sequence is fused to the C-terminus of each of the nuclease or effector protein and integrase protein, and the fusion protein is purified. The single guide RNA of the synthetic targeting genomic locus of interest is incubated with nuclease / effector protein to form a ribonucleoprotein complex. With the plasmid transfection cells containing the fluorescent marker of the selectable neomycin resistance marker (NeoR) or the flanking left end (LE) and right end (RE) motif, reclaim 4-6 hours, and then electroporate with nuclease RNP and integrase protein. By counting G418 resistant colonies or fluorescence activated cell cytometry, plasmid integration into the genome is quantitatively performed. Genomic DNA is extracted 72 hours after electroporation, and used to prepare NGS libraries. By making genome fragmentation and preparing the amplicon and flanking DNA of the transposon marker for NGS library preparation to measure the off-target frequency. At least 40 different target sites are selected to test the activity of every kind of targeting system.

[0336] Example 9 - Activity of Targeted Nucleases

[0337] In situ expression and protein sequence analysis showed that some RNA-guided effectors are active nucleases. They contain predicted endonuclease-related domains (matching RuvC and HNH_endonuclease domains) and predicted HNH and RuvC catalytic residues ( Figure 4A ).

[0338] Using an in vitro expression system and in vitro transcribed RNA, candidate activities were tested with engineered single guide RNA sequences. Active proteins that successfully cleaved the library produced a band of approximately 170 bp in the gel.

[0339] Example 10 - Identification of transposons

[0340] When transposon contains one or more protein sequences with transposase and / or integrase function between the left end and the right end of transposon, it is predicted that the transposon is active. As defined herein, the Tn7 transposon can include catalytic transposase TnsB, but can also contain TnsA, TnsC, TnsD, TnsE, TniQ and / or other transposases or integrases. The transposon end includes a predicted transposase binding site, which contains a direct and / or inverted repeat sequence of 15bp to 150bp in length, a side-joined transposase protein and other "goods" genes. Protein sequence analysis indicates that transposase contains an integrase domain, a transposase domain and / or a transposase catalytic residue, indicating that they are active (for example, Figure 4A and Figure 5 Figure A).

[0341] Example 11 - Identification of CRISPR-associated transposons

[0342] Putative CRISPR-associated transposons (CASTs) contain DNA and / or RNA targeting CRISPR effectors and proteins with predicted transposase function in the vicinity of the CRISPR array. In some systems, effectors are predicted to have nuclease activity (e.g., Figure 4A When the CRISPR locus (CRISPR nuclease and array) and the transposase protein are located between the left and right ends of the predicted transposon, the transposase is predicted to be associated with an active nuclease (e.g., Figure 4B -4C). In this case, effectors are predicted to direct DNA integration to a specific genomic location based on the guide RNA.

[0343] In some systems, effectors were predicted to have homology to documented CRISPR effector proteins but were inactive based on the absence of the endonuclease domain and / or catalytic residues ( Figure 5 When the CRISPR locus (inactive CRISPR nuclease and array) and the transposase protein are located within the left and right ends of the predicted transposon, the transposase is predicted to be associated with the effector ( Figure 5 Figure 5.2 shows the relationship between the two images.

[0344] Example 12-CAST Identification

[0345] CRISPR-associated transposons (CASTs) are a system that includes transposons that have evolved to interact with the CRISPR system to facilitate targeted integration of DNA cargo.

[0346] CAST is the genome sequence of one or more protein sequences of encoding, and described protein sequence relates to the DNA transposition in the characteristic left end and right end of transposon.As defined in this, Tn7 transposon can comprise catalytic transposase TnsB, but can also contain catalytic transposase TnsA, loading protein TnsC or TniB and target recognition protein TnsD, TnsE, TniQ and / or other transposon related components.The transposon end comprises the prediction transposase binding site, and it contains the direct and / or inverted repeat sequence that length is 15bp to 150bp, side joint transposon mechanism and other " goods " gene.

[0347] In addition, CAST also encodes DNA and / or RNA of CRISPR nucleases or effectors targeting the vicinity of the CRISPR array. In some systems, the effector is predicted to be an active nuclease based on the presence of a catalytic domain and / or catalytic residues associated with the nuclease. In some systems, the effector is predicted to have sequence similarity to the recorded CRISPR effector protein, but is inactive based on the absence of the nuclease domain and / or catalytic residue. When the CRISPR locus and transposon-associated proteins are located within the left and right ends of the predicted transposon, the transposon is predicted to be associated with the effector. In this case, the effector is predicted to guide DNA integration to a specific genomic location based on the guide RNA.

[0348] Example 13-2 Class Cas12K CAST

[0349] The Cas12k CAST system encodes a nuclease-deficient CRISPR Cas12k effector, a CRISPR array, tracrRNA, and a Tn7-like transposase protein. The Cas12k effector is phylogenetically distinct, and several features have been demonstrated to characterize its association with CAST ( Figure 8 For example, the left end of the transposon was identified downstream of the MG64-3 CRISPR locus, as indicated by the terminal inverted repeat sequence and the self-matching spacer sequence ( Fig.11A ). The Cas12k CAST CRISPR repeat sequence (crRNA) contains the conserved motif 5'-GNNGGNNTGAAAG-3' ( Fig. 9 ). The short repeat-antirepeat sequence (RAR) within the crRNA motif is aligned with different regions of tracrRNA ( Fig. 9 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), ( Fig. 10A ).

[0350] Example 14 - Transposon end prediction

[0351] Transposon ends were estimated from intergenic regions flanking effectors and transposon machinery. For example, for Cas12kCAST, the intergenic region immediately upstream of TnsB and downstream of the CRISPR locus was predicted to contain the left and right ends (LE and RE) of the Tn7 transposon.

[0352] Direct and inverted repeats (DR / IR) of ~12 bp were predicted on the contigs with up to 2 mismatches. In addition, a dot plot algorithm was used to find short (~10-20 bp) DR / IRs flanking the CAST transposon. Matching DR / IRs located in intergenic regions flanking the CAST effector and transposon genes were predicted to encode transposon binding sites. LEs and REs extracted from intergenic regions encoding putative transposon binding sites were aligned to define transposon end boundaries. Putative transposon LE and RE ends are regions that: a) are located within 400 bp upstream and downstream of the first predicted transposon encoding gene and the last predicted transposon encoding gene; b) share multiple short inverted repeats; and c) share >65% of nucleotide ids.

[0353] Example 15 - Single Guide Design

[0354] Analysis of the intergenic regions surrounding the Cas effectors and CRISPR array identified potential anti-repeat sequences and a conserved “CYCC(n6)GGRG” stem-loop structure adjacent to the anti-repeat sequence corresponding to the tracrRNA duplex sequence ( Fig. 11B ). TracrRNA and crRNA repeat sequences are folded and trimmed, and a GAAA tetraloop sequence is added to maintain the stem-loop region of the crRNA-tracrRNA complementary sequence.

[0355] Example 16 - In vitro integration activity using targeted nucleases

[0356] In situ expression and protein sequence analysis indicate 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. Using an in vitro expression system and in vitro transcribed RNA, candidate activity was tested with an engineered single guide RNA sequence. The active protein of the successful cutting library produced a band of about 170bp in the gel.

[0357] Example 17 - Programmable DNA Integration

[0358] CAST activity was tested with five types of components: (1) Cas effector protein expressed in an in vitro expression system (SEQ ID NO: 1); (2) target DNA fragment or plasmid containing the target sequence and the PAM corresponding to the Cas enzyme (SEQ ID NO: 31); (3) donor DNA fragment containing DNA markers or fragments flanked by LE and RE of the transposase system 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 sequence (SEQ ID NO: 5). The activity of the donor fragment that was successfully transposed was determined by PCR amplification of the donor-target junction.

[0359] After the transposition reaction was performed, PCR amplification of the junction showed that proper donor-target formation had occurred and that the transposition reaction was sg-dependent. Fig. 9 ). PCR amplifications for reactions #3 and #4 indicated two orientations for the donor relative to the target: one in which the LE was closer to the PAM, and one in which the RE was closer to the PAM. Although both transposition orientations were performed, there appeared to be a preference for donor integration in targets in which the LE was closer to the PAM, as indicated by the presence of strong bands for reactions #4 and #5.

[0360] Sanger sequencing of the preferred oriented products was performed. In integrations that occurred with the LE closer to the PAM, the sequencing chromatogram signal was clearly attenuated from either the forward or reverse orientation at the target / donor junction. This suggests that in products oriented with the LE closer to the PAM, integration occurred over a range of nucleotides, with the major product of the LE product closer to the PAM integrated as 61 bp from the PAM ( Fig. 10A Donor-derived sequencing at the donor-target junction defines the composition of the basic universe of LE and RE sequences ( Fig. 10A , Fig. 10B ). Sequencing of the RE on the LE product closer to the PAM showed a 3 bp duplication downstream of the donor RE ( Fig. 10B ). This is due in part to Tn7 transposase integration events that cleave and ligate the donor fragment at staggered cleavage sites. The 3 bp repeat is smaller than the expected 5 bp repeat from other Tn7 transposases.

[0361] Sanger sequencing of PCR amplification products on the 8N library of the target plasmid also indicated the PAM preference of the MG64-1 effector as nGTn / nGTt on the 5' end of the spacer (Figure 10C).NGS analysis of the PAM library targets confirmed the nGTn motif selectivity at the 5' end.

[0362] Further development of the single guide assay confirmed the activity of MG64-1 with the new sgRNA scaffold ( FIG. 13 ).

[0363] Example 18 - Integration Window Determination

[0364] The PCR junction of the amplified PAM was indexed and sequenced by NGS libraries. Reads were mapped and quantified using CRISPResso using the amplicon sequence of the putative transposition sequence at a 60 bp integration distance from the PAM (guide sequence = 3' end of 20 bp LE or RE, window center = 0, window size = 20). Indel histograms were normalized to the total indel reads detected and the frequencies were plotted relative to the 60 bp reference sequence ( Fig.14 )

[0365] PCR reaction 5 (LE proximal to PAM, Fig.14 Top panel) and PCR 4 (PAM-distal RE, Fig.14 Bottom panel) are plotted against the sequence of MG64-1 and the distance from the PAM. Analysis of the integration window shows that 95% of integrations occurring at the spacer PAM site are within a 10 bp window between 58 and 68 nucleotides from the PAM. The difference in integration distance between distal and proximal frequencies reflects integration site duplications - 3-5 base pair duplications caused by the staggered nuclease activity of the transposase during integration.

[0366] Example 19 - Colony PCR Screening for Transposase Activity

[0367] Transposition activity was determined by colony PCR screening. After transformation with the p donor plasmid, E. coli was plated on LB-agar containing ampicillin, chloramphenicol, and tetracycline. The selected CFU was added to a solution containing PCR reagents and primers flanking the selected insertion junction. The PCR reaction of the integrated product was visible on the gel ( Fig.15 ). Sequencing results of the selected colony PCR products confirmed that they represented a transposition event because the selected colony PCR products spanned the junction between the LE and PAM at the engineered target site in the lacZ gene ( Fig.16 ).

[0368] Example 20 - Single-Guide Engineering

[0369] The predicted RNA folds of active single RNA sequences were calculated at 37°C using the method of Andronescu 2007. All hairpin loop secondary structures were individually deleted from the construct and iteratively compiled into smaller single guides. Engineered single guides (esg) 4, 6, 7, 8, 9 were active for donor transposition ( Fig.17Panels C and D) where engineered sgRNAs 8 and 9 are weaker single guides and transposed using PCR 5 ( Fig.17 Engineered guide 5 was able to transpose, but engineered sgRNA 10 and PCR 5 were less transposed ( Fig.17 Panels 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 able to strongly transpose across both PCR 4 and 5 ( Fig.17 G and H), however, the combined addition of esg 6 and esg 18 to form esg19 resulted in a weaker transposition in PCR 5, and the addition of esg 7 to esg 19 to form esg 20 resulted in a very weak transposition junction in PCR 5 ( Fig.17 In the second approach, the MG64-1 tracrRNA was aligned with the recorded Vk-type tracrRNA, and the region of unique insertion was mutated to a single guide. The sgRNA was minimized by truncating the insert sequence of the MG64-1 sgRNA ( Fig.14 ). Two subsequent deletions, eg 2 and eg 3, were also tested ( Fig.17 Panels A and B), but neither esg 2 nor esg 3 caused significant transposition, so the single guide had a maximum reduction of 57 bases.

[0370] Example 21 - LE-RE Minimization

[0371] The sequencing of the target transposition junction helps to identify the terminal inverted repeat sequence by identifying the outermost sequence from the donor plasmid incorporated into the target reaction. By repeat sequence analysis of 14bp with a variability of 10%, the short repetitive sequence contained in the terminal was identified, and the truncation of these minimum ends was designed to retain the repetitive sequence while making the redundant sequence missing. Prediction and cloning are carried out in multiple iterations, and each interaction is tested by in vitro transposition. Initial LE and RE deletions are designed and cloned to 68bp, 86bp and 105bp of LE, 178bp, 196bp and 242bp of RE separately. The RE of 64-1 also has a significant sequence span without repetitive sequence, so the internal deletions of 50bp and 81bp were designed and cloned. The transposition between all single deletions is for PCR 4 and PCR 5 ( Fig.18 The 178, 196 and 212 bp trimmed ends of the former were cloned on the 81 bp internal deletion and tested for transposition. Transposition was active for all constructs designed. In combination with the 68 bp LE, transposition was demonstrated to be active when the 68 bp LE region was combined with the 96 bp RE region ( Fig.18 Panels E and F).

[0372] Example 22 - Effects of overhangs on transposition

[0373] In order to test whether the extra sequence outside the TnsB binding motif is necessary for transposition, oligonucleotides designed for the TGTACA motif of both LE and RE were designed and synthesized with 0, 1, 2, 3, 5 and 10 bp extra base pairs. These synthetic oligonucleotides are used to generate donor PCR fragments with overhangs and test their ability to transpose into the target site. Most notably, PCR 6 ( Fig.18 Panel G, lanes 1, 2), however, efficient integration at PCR 6 was detected with 0-3 bp overhangs, reflecting the RE that was not detected by the larger flanking sequences proximal to the PAM orientation.

[0374] Example 23-CAST NLS Design

[0375] Eukaryotic genome editing for therapeutic purposes relies heavily on importing editing enzymes into the nucleus. Small polypeptide segments of larger proteins signal cellular components for protein import across the nuclear membrane. Placing these tags is not an easy task because there is a potential trade-off between the import function and the function of the fused protein, depending on the position of the NLS tag. In order to test the functional orientation of NLS to each component of the CAST complex, constructs were designed and synthesized in which the nucleoplasmic protein NLS was fused to the N-terminus of each component of MG CAST and the SV40 NLS was fused to the C-terminus. The proteins of 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. Using PCR 4 (evaluation of RE distal transposition) and homologous transposition event PCR 5 (LE to proximal transposition), the activity maintenance of NLS-tagged constructs was evaluated by PCR at the donor-target junction.

[0376] Most components produce a single NLS orientation that maintains activity. TnsB is a CAST component active against both the N-terminal NLS and the C-terminal NLS by both PCR 4 and PCR 5 ( Fig.19 Panels A and B). TniQ is active against N-terminal NLS tags ( Fig.19 Panels C and D). And the Cas12k component is tagged with a C-terminal NLS ( Fig.19 Panels E and F, lanes 5, 6) were active. Further development of Cas12k with both nucleoplasmin and SV40 NLS tags was tested and found to be active ( Fig.19 Panels I and J, lane 4). TnsC binds to the N-terminal NLS ( Fig.19 Panels E and F, lane 7) had weak activity, but further exploration of TnsC tagging identified new working NLS-HA-TnsC and NLS-FLAG-TnsC constructs ( Fig.19 The end result is a fully NLS-tagged set of components that is active in vitro against both NLS-TnsB and TnsB-NLS orientations ( Fig. 20 Panels A and B, lanes 5 and 6).

[0377] Example 24-Cas12k and TniQ protein fusion construct design and testing

[0378] To simplify the expression of protein components and minimize the delivery of these components to cells, fusion constructs were designed, synthesized, and tested between the Cas12k effector and the TniQ protein. Two orientations of TniQ fused to Cas12k were designed and synthesized, C-terminal fusion Cas-TniQ and N-terminal fusion TniQ-Cas. Although both constructs were effective for PCR 4 ( Fig.21 Panel A) has weak activity, but when expressed in vitro and assayed for transposition ability, the PCR 5 junction is robustly formed by the TniQ-Cas fusion protein ( Fig.21 Figure 2. Transposition lengths determined using variable linker domains containing the original (20 amino acid linker), 48, 68, 72, and 77 amino acids. Fig.21 The NLS tag was then attached to the N-terminus of TniQ and the C-terminus of Cas12k, and it was found to be still active by PCR 5 ( Fig.21 Panels E and F).

[0379] Two other linkers were used to fuse the effector and TniQ genes. P2A, a self-stop translation sequence, was used in the Cas-NLS-P2A-NLS-TniQ construct ( Fig.21 Panels G and H, lane 6) and the MCV internal ribosome entry sequence (IRES) mRNA-based linker, which allows the two components to be translated independently in cells ( Fig.23 F and G).

[0380] Example 25 - Intracellular expression coupled with in vitro transposition assay

[0381] To test the function of the NLS construct in a physiologically relevant environment, constructs cloned with active NLS-tagged CAST components were integrated into K562 cells using lentiviral transduction. In short, the constructs cloned into the lentiviral transfer plasmid were transfected into 293T cells with encapsulation and packaging plasmids, and the virus containing the supernatant was collected from the culture medium after 72 hours of incubation. The culture medium containing the virus was then incubated with the K562 cell line and 8 μg / mL polybrene for 72 hours, and then the transfected cells were selected and batch integration was performed using 1 μg / mL of puromycin for 4 days. The selected cell lines were collected at the end of 4 days, and differential lysis was performed for nuclear and cytoplasmic fractions. The transposition ability of subsequent fractions and a set of complementary in vitro expression components was then tested.

[0382] Both NLS-TnsB and TnsB-NLS were tested by cell fractionation and in vitro transposition, and transposition was detected across both cytoplasmic and nuclear fractions, and NLS-TniQ had detectable activity in the cytoplasm ( Fig. 22 When expressed, NLS-HA-TnsC and NLS-FLAG-TnsC were active in both the cytoplasmic and nuclear fractions ( Fig. 22 Panel D), however, PCR4 was formed in the nuclear fraction of both TnsC constructs. Fig. 22 (Figure C).

[0383] When both NLS-TnsB or TnsB-NLS were linked to NLS-FLAG-TnsC using IRES, NLS-TnsB-IRES-NLS-FLAG-TnsC was mostly active in the nuclear fraction, while TnsB-NLS-IRES-NLS-FLAG-TnsC was active in both the cytoplasmic and nuclear fractions. This indicates that NLS-TnsB has a higher transport capacity to the nucleus ( Fig.21 Panels E and F).

[0384] Cas12k fusions in cells were similarly graded and tested for transposition. Cas-NLS Cas-NLS-P2A-NLS-TniQ was transduced into cells, graded and tested for subcellular activity in vitro. Cas-NLS-P2A-NLS-TniQ was able to transpose in the cytoplasm by adding a single guide to the reaction ( Fig.23 Panel A). The Cas-NLS-P2A-NLS-TniQ construct in the nuclear fraction was supplemented by supplementing the holo Cas protein (+sgRNA) or additional TniQ with sgRNA. This indicates that both Cas-NLS and NLS-TniQ made it to the nucleus ( Fig.23NLS-TniQ-Cas-NLS fusion protein has similar results, but requires TniQ ( Fig.23 D and E), and Cas-NLS-IRES-NLS-TniQ only requires complementation from holo Cas-NLS ( Fig.23 Panels F and G). Overall, this indicates that all components of CAST are able to be delivered to the nuclear fraction of cells.

[0385] Example 26 - Transposon end verification by gel shift

[0386] To verify the activity of TnsB on predicted transposon end sequences, the LE of MG64-1 was amplified using FAM-labeled oligonucleotides. The MG64-1 TnsB protein was expressed using a cell-free transcription / translation system and incubated with the LE FAM-labeled product. After 30 minutes of incubation, binding was observed on a native 5% TBE gel ( Fig.24 ). Co-incubation lanes ( Fig.24 , Lane 3) The multiple bands of fluorescent products indicate at least 2 TnsB binding sites.

[0387] The systems disclosed herein can be used for various 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 a subject's disease; inactivate a gene to determine its function in a cell; as a diagnostic tool for detecting pathogenic genetic elements (e.g., by cleaving retroviral RNA or amplified DNA sequences encoding pathogenic mutations); as an inactivating enzyme combined with a probe to target and detect specific nucleotide sequences (e.g., sequences encoding bacterial antibiotic resistance); inactivate a virus or make it unable to infect a host cell by targeting the viral genome; add genes or modify metabolic pathways to engineer an organism to produce valuable small molecules, macromolecules, or secondary metabolites; establish gene drive elements for evolutionary selection and / or as a biosensor to detect interference of foreign small molecules and nucleotides with cells.

[0388] Example 27-2 Cas12k CAST system prediction

[0389] The Cas12k CAST system encodes a nuclease-deficient CRISPR Cas12k effector, a CRISPR array, tracrRNA, and a Tn5053-like transposase protein ( Fig.25A ). The Cas12k effector is phylogenetically distinct and its association with CAST has been characterized ( Figures 25A-25BFor example, the left end of the transposon was identified downstream of many Cas12k effectors and their CRISPR loci, as indicated by the terminal inverted repeat sequence and the self-matching spacer sequence ( Figures 25A-25B ).

[0390] The transposon end of Cas12k CAST system is determined by the intergenic region of the flanking CRISPR locus and transposon mechanism. For example, the intergenic region directly located in the upstream of TnsB and the downstream of the CRISPR locus is predicted to contain the left and right ends of the transposon (LE and RE). These intergenic regions are compared between several homologs, and the conserved region is used to predict the transposon end boundaries (Figure 26).

[0391] The 3' end of the Cas12k CAST CRISPR repeat sequence (crRNA) contains a conserved motif 5'-GNNGGNNTGAAAG-3' when aligned between homologs, and they are predicted to bind to different regions of tracrRNA to form secondary and tertiary guide RNA structures ( Fig. 27 and Fig.28 Self-matching spacers within CAST transposons are often found next to pseudo-CRISPR repeat sequences near CRISPR arrays ( Fig.25A , bottom comparison).

[0392] Analysis of the intergenic regions surrounding the Cas effectors and CRISPR array identified potential anti-repeat sequences and a conserved “CCYCC(n6)GGRGG” stem-loop structure adjacent to the anti-repeat sequence corresponding to the tracrRNA duplex sequence ( Fig. 27 ). High-quality tracrRNA was used to construct the covariance model and searched for all Cas12k CAST genomic fragments identified in this study.

[0393] For single guide RNA (sgRNA) design, tracrRNA and crRNA repeat sequences were folded and trimmed, and a GAAA tetraloop sequence was added to maintain the stem-loop region of the crRNA-tracrRNA complementary sequence ( Fig.28 Typically, sgRNAs share conserved structural features but share less than 70% pairwise nucleotide identity ( Fig. 27 ).

[0394] Example 28-In vitro characterization of the Cas12k CAST system

[0395] In order to test the function of the Cas12k CAST system and to illustrate potential PAM, the transposition reaction was assembled using a synthetic Cas12k effector and Tn5053-like protein under the control of a T7 promoter. Each open reading frame was expressed in vitro by an in vitro expression system, and in the case of an 8N target library, a transposition buffer, a donor PCR fragment, and a target based on a plasmid were assembled in the transposition reaction (a small figure A of Figure 29). When the CAST system is active and the donor fragment can be transposed into the target plasmid library, the transposition reaction can be amplified by PCR to recover each donor-target junction of two potential transposition products (a small figure B of Figure 29).

[0396] Among the discovered Cas12k CAST systems, three were prioritized for their novelty and completeness and tested for their transposition potential in vitro. MG64-1 CAST was able to transpose cargo into a donor plasmid in an sgRNA-dependent manner ( Fig. 30A ). For each of the potential junction PCR amplifications, it was predicted that all four junctions would be observed if both orientations of integration were complete, or only two of the junctions would be observed if integration only occurred in a single orientation (Figure 29, panel B). Surprisingly, robust transposition was observed in three of the four junction PCR reactions. The observed reactions represented two potential left-end junction products for the target-LE-cargo-RE (T-LR) and target-RE-cargo-LE (T-RL) orientations (PCR 5 and PCR 3, respectively) and a right-end product for the T-LR orientation (PCR 4) ( Fig. 30A ).

[0397] Sanger-based sequencing of PCR transposition fragments accurately aligns to the target sequence, and the sequencing signal decays rapidly at the target-donor junction ( Fig.41 ). Signal decay indicated the integration product population and sequencing from the donor end of the transposition product verified the LE and RE predictions of the MG64-1 system.

[0398] To elucidate the PAM preference of the active CAST system, successful transposition events from a population of 8N random libraries that received donor sequences were sequenced by NGS. Sequencing reads identified the GTN-5' PAM ( Fig. 30B ).

[0399] In addition to identifying the PAM, it is also helpful to determine the integration window size: the relative distance from the PAM at which integration occurs. Cas12k CAST integrates cargo at a specific window of 60 bp away from the PAM motif. When NGS reads of the transposition junction of MG64-1 were quantified, it was determined that 99% of the integration events occurred between 57 and 67 base pairs away from the PAM ( Fig. 30C In addition, the shift in distal distance indicates the presence of target site duplications (TSDs). For MG64-1, the TSD window ranged from 3 to 5 bp, which is consistent with the 4 bp TSD observed in the metagenomic assembly ( Fig.25A ).

[0400] Example 29-Single guide RNA engineering

[0401] While nuclease-dead effectors of CAST are typically less than 700 aa, tracrRNAs are large. Multiple sequence alignment of predicted tracrRNAs indicated that MG64-1 tracrRNA contained a large 58 bp insertion ( Fig. 27 ). When folded, the excess sequence forms two halves of a well-predicted hairpin near the GCGC pseudoknot motif ( Fig.28 and Fig.31 ). It was hypothesized that although these structures were well predicted to form hairpin loops, if this additional sequence was missing, the conservation of the surrounding sequence would help stabilize this region. Therefore, a series of truncations were made to the backbone of this region including the single guide, including a larger truncation of the GCGC pseudoknot ( Fig.31 The single guide RNA containing the most conserved deletion is the only functional form of the single guide ( Fig.31 and Fig.28 , esg1). Subsequent studies of deletions at both the 5' and 3' ends of the single guide showed that the crRNA::tracrRNA repeat anti-repeat sequence hairpin is also dispensable for function, whereas the use of base nucleotide pairing is ( Fig.31 ). The combined recorded functional deletions largely abolished activity. Thus, the minimal functionally engineered 64-1 guide was 270 nt ( Fig. 27 , esg1 4a).

[0402] While minimizing the number of guides, the cross-functional potential of the minimal single guides predicted for MG64-2, MG64-3, and MG64-5 was tested using the MG64-1 CAST system. It was observed that the MG64-2 single guide was active for the MG64-1 CAST system, thus reducing the functional size of the sgRNA to 251 ( Fig.32 ). The PAM requirements of the MG64-1 CAST with the 64-2 single guide were no different from effectors with the native single guide.

[0403] To further reduce the size of the MG64-2 sgRNA, a series of truncations were designed and tested to determine whether 64-1CAST could continue to utilize a cross-functional single guide for targeted integration ( Fig.33Six deletions were tested across the MG64-2 single guide, covering combinations of truncation of the 5' pseudoknot, internal deletions at the terminal hairpin, and deletion of the CRISPR repeat anti-repeat sequence hairpin. Two engineered sgRNAs containing deletion of the 5' pseudoknot and shorter length hairpin 3 maintained the ability to transpose with MG64-1 ( Fig.34 ).

[0404] To simplify the synthesis of large RNA molecules, the sgRNA was split into two fragments that can be recombined during the transposition reaction. This approach uses a structured hairpin loop to recombinate two RNA molecules together in order to form a structure similar to that of an active sgRNA. Using the MG64-2 single guide, five split points were designed between the predicted main chain helix, unstructured regions, and highly predicted hairpin loops ( Fig.35 ). In addition, one segment contains an extension that improves base pairing of the segmented hairpin loop. In the designs tested, the hairpin loop representation and the extended hairpin dual guide are able to complex with Cas12k of the MG64-1 system and guide transposition ( Fig.36 ).

[0405] Example 30 - Transposon LE and RE Engineering

[0406] The intergenic region flanking the Cas12k CAST transposon genes TnsB, TniB, TniQ, and Cas12k contains ~12-20bp of terminal inverted repeats (TDRs), which are predicted to encode transposon binding sites. Since the TIRs on the LE and RE are predicted to be TnsB binding sites, a series of deletions at the wild-type LE and RE ends were generated to determine the necessary binding sites. Although the LE and RE of MG64-1 are predicted to contain 3 and 5 repeat sequences, respectively, the deletion of the TIR cargo end maintains activity to fragments as small as 68bp. The RE deletion series method contains an internal deletion of 81bp, which may allow the size of the RE to be reduced to include 4 repeat sequences, up to 97bp. Minimization of LE and RE maintains the transposition activity of the system ( Fig.37 ).

[0407] Example 31 - Integration activity of E. coli and MG64-1

[0408] To test transposition efficiency in a cellular environment, the E. coli BL21 (DE3) strain was engineered to contain a spacer sequence with demonstrated in vitro activity. A plasmid containing a polycistronic Tn5053-like gene and effectors under a T7 promoter was used to express the CAST protein, and a separate plasmid was co-transformed to introduce the guide under the control of the J23119 promoter ( Fig.38A ). The p donor plasmid contained the antibiotic resistance cargo flanked by the confirmed WT LE and RE and the minimized LE and RE of MG64-1.

[0409] An NGS-based method was developed to assess the transposition efficiency of MG64-1. NGS reads indicated over 80% editing efficiency ( Fig.38B ), and were able to determine the off-target profile associated with each CAST. The off-target editing rate was determined as a single read mapping to an LE or RE with an additional 14 bases mapping elsewhere in the E. coli genome. No off-target integrations greater than 1% of all total transposition events were detected ( Fig.38C ).

[0410] Example 32 - Endogenous locus targeting

[0411] To test the programmability of these systems for integration into the E. coli genome, three target sites with a GTN-5' PAM were selected for integration. Based on WGS data, MG64-1 CAST was able to integrate at multiple loci with efficiencies ranging between 50-90% ( Fig.39 Together with the low off-target rates, these data suggest that this Cas12k system is capable of achieving high genomic integration rates with programmable RNA guides.

[0412] Example 33 - Multi-locus targeting

[0413] With high levels of integration at both the endogenous locus and the engineered locus, CAST was then tested for its ability to integrate into multiple copies in a single reaction. By introducing two single guides along with a donor plasmid into a single E. coli strain, both loci were shown to integrate with greater than 50% efficiency (Figure 40). In all cases, the combination of integration at both loci accounted for more than 95% of all integrations occurring on the genome (Figure 41). Fig.40A ).

[0414] Example 34 - Exploration of Target DNA Dilutions in In Vitro Transposition Assays

[0415] In vitro targeted integrase activity assay

[0416] Integrase activity was measured using a target plasmid (ptarget) containing a PAM adjacent to the protospacer sequence ( Fig.42A All transposase, single guide RNA (sgRNA), and effector components were amplified by PCR into the T7 promoter leader gene sequence and expressed independently in an in vitro transcription / translation system ( Fig.42A). Purified in vitro transcribed single guide RNAs were refolded in duplex buffer (10 mM Tris pH 7.0, 150 mM NaCl, 1 mM MgCl2) and normalized to 1 μM. Donor fragments were PCR amplified from plasmid pDonor, which contains kanamycin or tetracycline resistance markers flanking the MG64-1 left (LE) and right (RE) transposon motifs, and normalized to 50 ng / μL.

[0417] After expression, 1 μL of Cas12k in vitro expression reaction was added to 0.5 pmoles of sgRNA and incubated at 25°C for 20 minutes. The transposase protein expressed alone was then added in 1 μL volume for each expression. The target DNA and 50 ng of donor DNA were then added to the transposition reaction in the reaction buffer, with a final concentration of 26 mM HEPES pH 7.5, 4.2 mMTris pH 8, 50 μg / mL BSA, 2 mM ATP, 2.1 mM TCEP, 0.05 mM EDTA, 0.2 mM MgCl2, 28 mM NaCl, 21 mM KCl, 1.35% glycerol (final pH 7.5) and 15 mM Mg (OAc) 2. The in vitro transposition reaction was performed at 37°C for 2 hours, the transposition reaction was diluted tenfold in water, and then used as a template for junction PCR analysis.

[0418] Junction PCR analysis

[0419] The junction PCR reaction was performed with Q5 polymerase and amplified with primers flanking: Rxn#1 (target), Rxn#2 ​​(donor), Rxn#3 (reverse LE), Rxn#4 (forward RE), Rxn#5 (forward LE), and Rxn#6 (reverse RE) ( Fig.42B ). PCR fragments were run on 2% agarose gels in 1x TAE and analyzed for size discrimination. The appropriate size bands of each PCR junction were gel excised and the PCR fragments were recovered by purification and Sanger sequencing using two amplification primers. The resulting Sanger sequencing was mapped to the donor and target sequences to confirm integration of approximately 60 bp from the PAM.

[0420] Transposition reaction with target plasmid DNA dilution

[0421] Given the known low availability of single-copy target sites in the human genome (e.g., Moreb et al., 2020), it was predicted that low-availability targets in in vitro experiments could be mimicked by the search space in human genomic DNA using MG64-1 CAST. To determine the minimum amount of target plasmid (ptarget) required for in vitro detection of MG64-1 targeting, total target DNA was serially diluted using CAST in transposition reactions. The target plasmid amount was serially diluted 10-fold from 50 ng DNA (molar) per reaction to 0.00005 ng (molar) and then added to the transposition reaction containing the MG64-1 kit, sgRNA, and donor plasmid ( Fig.42A ). PCR amplification of the target-donor junction across the transposition products was then analyzed by gel electrophoresis ( Fig.42B ).

[0422] Results: Serial dilution of target plasmid DNA

[0423] In vitro transposition experiments with target plasmid dilutions indicated that targeted integration of the donor into the plasmid required a single guide RNA (sgRNA) ( Fig.42C , lanes 1 and 2). When the target was serially diluted, the target DNA was still detectable at 0.00005 ng / reaction, but at a lower intensity ( Fig.42C , "target" band on lane 8). Both the reverse LE integration product (Rxn#3) and the reverse RE product (Rxn#6) decreased with ten-fold dilution, resulting in the detectable presence of Rxn#3 as low as 0.5 ng target DNA ( Fig.42C ). The transposition reactions of Rxn#4 and #5 were equally robust and could detect as little as 0.05 ng of target DNA.

[0424] Based on the presence of three of the four expected transposition reaction products, the 0.5 ng target plasmid condition was chosen to test whether transposition was detected when increasing the complexity of the DNA search space. Increasing amounts of exogenous human genomic DNA (gDNA) were added to the reaction with fixed 0.5 ng target plasmid, MG64-1 CAST, and sgRNA ( Fig.42A and Fig.42D ). When no gDNA was added, transposition experiments confirmed that sgRNA was required for targeted integration of the donor into the target plasmid at 0.5 ng DNA ( Fig.42D , lanes 1 and 2). When gDNA was added to the reaction in increasing amounts (0-2000 ng DNA per reaction), the transposition product was also diluted, as indicated by the faint bands compared to the no gDNA control ( Fig.42DFor example, the reverse LE integration product (Rxn#3) was visible when up to 50 ng of gDNA was added. In addition, robust transposition products were detected for the forward RE (Rxn#4) and forward LE (Rxn#5) products at 125 to 1000 ng of gDNA and a fixed amount of 0.5 ng of target plasmid. Fig.42D , lanes 4-8).

[0425] Example 35 - In vitro transposition into human gDNA using MG64-1 CAST

[0426] In vitro targeting of high-copy elements across the human genome

[0427] This example evaluates a dilution series of a natural target in the genome for transposition as a function of target frequency.

[0428] Results: Target site identification in high-copy regions of the human genome

[0429] Identification of high-copy targets in the human genome using Cas off-target finders. Using 200-300 bp target sequences in the most conserved space of each repetitive element, 15 target sites were identified for each LINE1 3' and HERV, and 7 target sites were designed for SVA elements with different GC content, orientation, and MG64-1 rGTN PAM substitutions ( Fig.43A ). The target (spacer) sequence was synthesized as an oligonucleotide and PCR amplified onto the MG64-1 sgRNA template with a T7 promoter upstream of the single guide backbone. The PCR reaction of the MG64-1 sgRNA was then purified and transcribed in vitro. Using the NLS-tagged MG64-1 protein components, an in vitro transposition reaction as described above was assembled with 1 μg / reaction of purified HEK293T gDNA as the target DNA.

[0430] Results: In vitro targeted transposition into high-copy regions of the human genome

[0431] Successful transposition of MG64-1 was assessed by the resulting forward PCR and reverse PCR junction products at each of the 15 target sites in the high copy element ( Fig.43B MG64-1 promotes transposition to LINE1 targets 3, 5, 6, 7, 10, and 13 in the forward orientation, whereas targets 1, 2, 8, 9, 12, 14, and 15 are reactive to transposition in both the forward and reverse orientations ( Fig.43B In addition, active transposition to SVA target 3 and HERV target 5 was specific for both the forward and reverse orientations ( Figures 43C-43D ). Sanger sequencing of the transposition reaction products of LINE1, SVA, and HERV confirmed that the in vitro transposition was specific and RNA-guided ( Figures 43E-43H).

[0432] Example 36 - Fusion of the NLS functional domain with MG64-1 CAST can target high copy elements

[0433] Functional domains fused to CAST components for targeted transposition to high-copy elements in the human genome in vitro

[0434] In cases where experiments indicated that high copy elements were effective targets for in vitro integration, the effects of the functional domains on CAST targeting at these sites were tested. Functional domain fusions of Cas12k-sso7d were primed with H1core-TniQ or Cas12k-sso7d was primed with HMGN1-TniQ to transpose donor DNA in reaction with NLS-TnsB and NLS-TnsC fusions at high copy elements LINE1 target 12 and target 15. Where indicated, transposition reactions were assembled with Cas12k alone or fusion Cas12k-sso7d, with a no sgRNA (-sg) condition as a negative control for transposition, and where indicated, with sgRNAs for target 12 and target 15 of the LINE1 3' element ( Fig.44 ). In addition, the transposition reaction was supplemented with translated NLS-TniQ, NLS-H1 core-TniQ or NLS-HMGN1-TniQ, NLS-TnsB, NLS-TnsC, p donor, buffer and human gDNA for targeting.

[0435] result

[0436] In vitro transposition assays with fusion domains targeting LINE1 target 12 and target 15 indicated that MG64-1 no fusion, positive control ( Fig.44 , lanes 3 and 7) and the fusion Cas12k-sso7d successfully integrated with HMGN1-TniQ ( Fig.44 , lanes 5 and 9). When no sgRNA was added to the reaction, no targeting occurred (Figure 4, lanes 2 and 6).

[0437] Example 37 - NLS fused to S15 for targeted transposition

[0438] NLS fusion to the small ribosomal protein subunit S15 is essential for the correct orientation of the tag

[0439] Recently, the small prokaryotic ribosomal protein subunit S15 was considered to be required for in vitro Cas12k CAST-targeted transposition (Schmidt et al., 2022; Park et al., 2022). Therefore, the need for S15 with and without an NLS tag was evaluated in transposition experiments performed with MG64-1. Since the in vitro expression reagents already contain S15 (S15 is part of the prokaryotic ribosomal complex required for protein expression), in vitro transposition experiments performed with PURExpress-expressed CAST proteins may allow S15 to be carried over from the expression step and subsequently recruited for CAST targeting.

[0440] Results: S15 improves transposition efficiency in in vitro experiments

[0441] Wheat germ extract was used in a eukaryotic transcription / translation system without S15 to express the MG64-1 CAST component. The CAST template was amplified to contain a T7 promoter and a 40 bp Poly A tail for transcriptional stability of the mRNA template. As described above, proteins were expressed from dsDNA templates by transcription / translation reactions and then used in in vitro transposition reactions. The results indicated that S15 addition increased the efficiency of targeted transposition, as shown by the intensity of the band from the junction PCR product (Rxn#5) ( Fig.45A , lanes 4-5).

[0442] Results: S15-NLS fusion is the preferred orientation for in vitro transposition

[0443] Under eukaryotic conditions, protein translation occurs exclusively in the cytoplasm, whereas CAST-mediated transposition reactions most likely occur in the nucleus. The necessity of an NLS tag for nuclear localization of S15 was evaluated. The NLS tag was fused to both the N-terminus and the C-terminus of S15 and tested in eukaryotic in vitro transcription / translation reactions and in vitro transposition experiments ( Fig.45A , Lane 5 and Fig.45B , lanes 4 and 5). The results indicate that the transposition of S15-NLS is more efficient than the other tested conditions ( Fig.45A , lane 5).

[0444] Example 38-S15 Design of CAST vector essential for cellular translation of CAST

[0445] The MG64-1 CAST protein was expressed on two high expression plasmids for transposition experiments in human cells. One plasmid expressed the protein targeting complex under the control of the pCAG promoter. Two forms of the protein targeting complex were designed. One form contained a Cas12k-sso7d functional domain fusion in which the 2A peptide was fused to S15-NLS, IRES, and NLS-H1core-TniQ ( Fig.46A , top left). The second version contains Cas12k-sso7d-2A-S15-NLS with NLS-HMGN1-TniQ fusion ( Fig.46A , bottom left). The targeting plasmid also contains the pU6 PolIII promoter, which drives transcription of humanized MG64-1 sgRNAs for targeting LINE1 targets 8, 12, and 15 and one of the SVA targets 3. The second plasmid transfected into the cells is a donor plasmid containing NLS-TnsB and NLS-TnsC separated by an IRES under expression from the pCAG promoter. On this plasmid, a 2.5 kb DNA cargo ( Fig.46A , right side).

[0446] HEK293T lipid transfection

[0447] 2.5 million HEK293T cells were seeded 24 hours before lipid transfection of two plasmid systems with 9 μg: 9 μg targeting: donor plasmids. The cells were incubated at 37 ° C for 72 hours and then collected by resuspending in 4 mL 1x PBS pH 7.2. 2 mL of resuspended cells were collected for gDNA and eluted in 200 μL elution buffer. The transposition of 5 μL extracted gDNA in 100 μL Q5 PCR reactions was determined with primers specific to high copy element targets: transposition in the forward direction was determined by amplification with primers specific to forward PCR, and reverse transposition was determined by amplification with primers specific to reverse PCR. The amplified PCR reactions were visualized on 2% agarose gels. As observed in the in vitro transposition experiment, the transposition was predicted to transpose at 60 nt from the PAM, and activity was determined by the presence of a single band amplified by junction PCR at the predicted size. PCR amplicons were Sanger sequenced and sequenced by NGS for transposition profiling.

[0448] result

[0449] Analysis of transposition of cells with H1core-TniQ or HMGN1-TniQ transfected with two forms of the targeting complex plasmid ( Fig.46B Both forms of the targeting complex plasmid promoted transposition at all four target sites ( Fig.46B , arrows). LINE1 targets 8 and 15 were only detectable in the LE to 5' target orientation, while LINE1 target 12 was only detectable in the LE to 3' orientation ( Fig.46B ). The results indicate that targeted integration in human cells has a strong preference for directionality from the PAM.

[0450] Sanger sequencing of the PCR junctions confirmed integration at 8, 12, and 15 nt from the LINE1 target, 59, 62, and 60 nt from the PAM, respectively ( Figures 46C-46H ). Sequencing signal decay was observed at transposition junctions, which was caused by mixing of population events. To determine the single molecule profile of each integration event, PCR amplicons were sequenced by NGS. Reads generated by NGS sequencing confirmed targeted integration at LINE1 targets 8, 12, and 15 and SVA target 3. The variation in the target regions indicates the natural diversity of LINE1 and SVA repeat elements in the human genome.

[0451] Overall, MG64-1 is a successful system for targeted integration in human cells, with a strong directional preference relative to the PAM.

[0452] Example 39-NLS Immunofluorescence

[0453] Staining of fixed cells showed that all NLS-CAST components were able to translocate to the nucleus

[0454] While it is possible to test functional NLS-CAST fusions using in vitro transposition assays, the intrinsic ability of the NLS tag to shuttle the protein into the nucleus must be tested in a cellular context. By exposing the CAST-NLS protein in cells, either through genomic expression or in the form of mRNA, the cells can be fixed and stained for the introduced epitope tag using a fluorescently conjugated antibody. By visualizing the fluorescence relative to the DAPI nuclear stain, the potential for the protein to translocate into the nucleus to exert activity can be determined.

[0455] Immunofluorescence in HEK293T cells

[0456] HEK293T cells were plated on collagen-coated coverslips at 50,000 cells / 24-well plate. Cell cultures were allowed to adhere to coverslips overnight. For each of the active MG64-1 NLS fusion proteins, the template was transcribed in vitro with a poly-A tail, and the mRNA of these constructs was transfected in HEK293T at 500ng / well. After 48 hours of expression, cells were fixed with 4% formaldehyde, cell membranes were permeabilized with Triton X-100, then washed with 2% BSA and probed with anti-HA antibody overnight. The cells were then washed with 2% BSA / PBS and then stained with FITC-conjugated goat anti-mouse secondary antibodies. After secondary antibody exposure, cells were washed with PBS, mounted on DAPI mounting epoxy, and cured overnight. Cells were fluorescently visualized on a cell imaging microscope, and nuclear localization was determined by colocalization of FITC and DAPI staining.

[0457] result

[0458] Cas12k-NLS, NLS-TnsB and NLS-TniQ of MG64-1 CAST complex can be localized in the nucleoplasm ( Fig.47A , top and middle panels, and Fig.47B ), while NLS-TnsC is localized in the cytoplasm in filamentous aggregates ( Fig.47A , bottom panel). When NLS-TnsC was subsequently co-expressed with NLS-TniQ for immunofluorescence studies, the nuclear localization of NLS-TnsC was rescued ( Fig.47C ). In addition, when all MG64-1 Cas12k CAST components were expressed in HEK293T cells via mRNA delivery, all components were localized in the nucleus ( Fig.47D ). The results indicate that the filamentous aggregation of NLS-TnsC in cells can be abolished by the presence of TniQ in cells.

[0459] Example 40 - In vitro transposition using purified MG64-1 targeting complex

[0460] Construct design...

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 2 type V Cas effectors, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide configured to hybridize to the target nucleic acid site; b) a Tn7-type transposase complex configured to bind to the Cas effector complex and comprising TnsB, TnsC and TniQ components; as well as c) a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex and comprising the cargo nucleotide sequence.

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

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

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

5. The system according to any one of claims 1 to 4, 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.

6. The system of claim 5, wherein the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 9, 11, 36-38, 76, and 78.

7. The system of any one of claim 5, wherein the right recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93.

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

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

31.

10. The system of any one of claims 8 to 9, wherein the PAM sequence is located 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 3' to the target nucleic acid site.

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

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

14. The system of any one of claims 1 to 12, wherein the Class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85 and 220.

15. The system of any one of claims 1 to 12, wherein the Class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85 and 220.

16. The system of any one of claims 1 to 12, wherein the class 2 type V Cas effector comprises a polypeptide comprising the sequence of any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85 and 220.

17. The system of any one of claims 1 to 16, wherein the TnsB component comprises a polypeptide having a sequence at least 80% identical to any one of SEQ ID NOs: 2, 13, 17 and 65.

18. The system of any one of claims 1 to 16, wherein the TnsB component comprises a polypeptide having a sequence at least 90% identical to any one of SEQ ID NOs: 2, 13, 17 and 65.

19. The system of any one of claims 1 to 16, wherein the TnsB component comprises a polypeptide having a sequence of any one of SEQ ID NOs: 2, 13, 17 and 65.

20. The system of any one of claims 1 to 19, wherein the Tn7-type 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 to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111.

21. The system of any one of claims 1 to 19, wherein the Tn7-type 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 to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111.

22. The system of any one of claims 1 to 19, wherein the Tn7-type 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 NOs: 3-4, 14-15, 18-19, 66-67 and 109-111.

23. The system of any one of claims 1 to 22, wherein the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222.

24. The system of any one of claims 1 to 22, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 106, 107, 108, 5, 45-63, 68-75, 96-103, and 123-140.

25. The system of any one of claims 1 to 23, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 187-189.

26. The system of 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 to any one of SEQ ID NOs: 181-183.

27. The system of any one of claims 1 to 26, wherein the class 2 type V Cas effector and the Tn7-type transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

28. 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 a class 2 type V Cas effector and an engineered guide polynucleotide configured to hybridize to the target nucleic acid site, wherein the Cas effector complex comprises a polypeptide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220; b) a Tn7-type transposase complex configured to bind to the Cas effector complex and comprising TnsB, TnsC, and TniQ components, wherein the TnsB, TnsC, or TniQ components comprise a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2-4, 13-15, 17-19, 65-67, and 109-111; and c) a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex and comprising the cargo nucleotide sequence.

29. The system of claim 28, wherein the Cas effector complex is non-covalently associated with the Tn7-type transposase complex.

30. The system of claim v, wherein the Cas effector complex is covalently linked to the Tn7-type transposase complex.

31. The system of claim 28, wherein the Cas effector complex is fused to the Tn7-type transposase complex.

32. The system of any one of claims 28 to 31, wherein the cargo nucleotide sequence is flanked by a left-side transposase recognition sequence and a right-side transposase recognition sequence recognized by the Tn7-type transposase complex.

33. The system of claim 32, wherein the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 9, 11, 36-38, 76, and 78.

34. The system of any one of claim 32, wherein the right recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93.

35. The system of claims 28 to 34, wherein the target nucleic acid comprises a PAM sequence that is compatible with the Cas effector complex.

36. The system of claim 35, wherein the PAM sequence comprises SEQ ID NO:

31.

37. The system of any one of claims 35 to 36, wherein the PAM sequence is located about 50 to about 70 base pairs from the target nucleic acid site.

38. The system of claim 37, wherein the PAM sequence is located 3' to the target nucleic acid site.

39. The system of claim 37, wherein the PAM sequence is located 5' to the target nucleic acid site.

40. The system of any one of claims 28 to 39, wherein the class 2 type V Cas effector is a Cas12k effector.

41. The system of any one of claims 28 to 39, wherein the Class 2 type V Cas effector comprises a polypeptide comprising a sequence at least 90% identical to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220.

42. The system of any one of claims 28 to 39, wherein the Class 2 type V Cas effector comprises a polypeptide comprising the sequence of any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220.

43. The system of any one of claims 28 to 39, wherein the TnsB, TnsC or TniQ component comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2-4, 13-15, 17-19, 65-67 and 109-111.

44. The system of any one of claims 28 to 39, wherein the TnsB, TnsC or TniQ component comprises the sequence of any one of SEQ ID NOs: 2-4, 13-15, 17-19, 65-67 and 109-111.

45. The system of any one of claims 28 to 44, wherein the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222.

46. ​​The system of any one of claims 28 to 44, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 106, 107, 108, 5, 45-63, 68-75, 96-103, and 123-140.

47. The system of any one of claims 28 to 46, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 187-189.

48. The system of any one of claims 28 to 46, wherein the small prokaryotic ribosomal protein subunit S15 is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 181-183.

49. The system of any one of claims 28 to 48, wherein the class 2 type V Cas effector and the Tn7-type transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

50. 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 configured to hybridize to the target nucleic acid site and comprising: i) 2 types of V-type Cas effectors, said 2 types of V-type Cas effectors comprising SEQ ID NO: 1, 81, 82, 83 and 85 having at least 80% sequence identity; and ii) an engineered guide polynucleotide comprising at least 80% identity to any one of SEQ ID NOs: 5, 6, 45-63, 68-75, 96-103, and 123-140; b) a Tn7-type transposase complex configured to bind to the Cas effector complex and comprising TnsB, TnsC, and TniQ components, wherein the TnsB, TnsC, or TniQ components comprise a sequence having at least 80% identity to any one of SEQ ID NOs: 2-4; and c) a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex and comprising, in 5' to 3' order: i) a left recombinase sequence comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9, 11, 36, 37, and 38; ii) the cargo nucleotide sequence; and ii) a right recombinase sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 8, 39-44 and 93.

51. 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 configured to hybridize to the target nucleic acid site and comprising: i) 2 types of V-type Cas effectors, said 2 types of V-type Cas effectors comprising SEQ ID NO: 12 sequences with at least 80% sequence identity; and iii) an engineered guide polynucleotide comprising at least 80% identity to any one of SEQ ID NOs: 32, 102, 104, and 107; b) a Tn7-type transposase complex configured to bind to the Cas effector complex and comprising TnsB, TnsC, and TniQ components, wherein the TnsB, TnsC, or TniQ components comprise a sequence having at least 80% identity to any one of SEQ ID NOs: 13-15; and c) a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex and comprising, in 5' to 3' order: i) a left recombinase sequence comprising a sequence having at least 80% sequence identity to SEQ ID NO: 76; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence, the right recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NO:

77.

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 configured to hybridize to the target nucleic acid site and comprising: i) 2 types of V-type Cas effectors, said 2 types of V-type Cas effectors comprising SEQ ID NO: 16 sequences having at least 80% sequence identity; and ii) an engineered guide polynucleotide comprising at least 80% identity to any one of SEQ ID NOs: 33, 103, 105, and 108; b) a Tn7-type transposase complex configured to bind to the Cas effector complex and comprising TnsB, TnsC, and TniQ components, wherein the TnsB, TnsC, or TniQ components comprise a sequence having at least 80% identity to any one of SEQ ID NOs: 17-19; and c) a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex and comprising, in 5' to 3' order: i) a left recombinase sequence comprising a sequence having at least 80% sequence identity to SEQ ID NO: 78; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence, the right recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NO:

79.

53. 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 configured to hybridize to the target nucleic acid site and comprising: i) 2 types of V-type Cas effectors, said 2 types of V-type Cas effectors comprising SEQ ID NO: 1, 81, 82, 83 and 85 having at least 80% sequence identity; and ii) an engineered guide polynucleotide comprising at least 80% identity to any one of SEQ ID NOs: 5, 6, 45-63, 68-75, 96-103, or 123-140; b) a Tn7-type transposase complex configured to bind to the Cas effector complex and comprising TnsB, TnsC, and TniQ components, wherein the TnsB, TnsC, or TniQ components comprise a sequence having at least 80% identity to any one of SEQ ID NOs: 2-4; and c) a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex and comprising, in 5' to 3' order: i) a left recombinase sequence comprising a sequence having at least 80% sequence identity to SEQ ID NOs: 9, 11, 36, 37, and 38; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NOs: 8, 39-44 and 93.

54. The system of any one of claims 50 to 53, further comprising a PAM sequence that is compatible with the Cas effector complex.

55. The system of claim 54, wherein the PAM sequence comprises SEQ ID NO:

31.

56. The system of any one of claims 54 to 55, wherein the PAM sequence is located about 50 to about 70 base pairs from the target nucleic acid site.

57. The system of claim 56, wherein the PAM sequence is located 3' to the target nucleic acid site.

58. The system of claim 56, wherein the PAM sequence is located 5' to the target nucleic acid site.

59. The system of any one of claims 50 to 58, wherein the Cas effector complex further comprises a small prokaryotic ribosomal protein subunit S15.

60. The system of claim 59, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 187-189.

61. The system of claim 59, wherein the small prokaryotic ribosomal protein subunit S15 is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 181-183.

62. 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 2 types of V-type Cas effectors, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide, wherein the engineered guide polynucleotide hybridizes to the target nucleic acid site; b) a Tn7-type transposase complex, which is operably linked to the Cas effector complex and includes TnsB, TnsC and TniQ components; as well as c) a double-stranded nucleic acid, which comprises, in 5' to 3' order: i) left recombinase recognition sequence; ii) the cargo nucleotide sequence; and iii) a right recombinase recognition sequence, wherein the left recombinase recognition sequence and the right recombinase recognition sequence are recognized by the Tn7-type transposase complex.

63. The system of claim 62, wherein the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 9, 11, 36-38, 76, and 78.

64. The system of any one of claim 62, wherein the right recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 8, 10, 39-44, 77, 79, and 93.

65. The system of claims 62 to 64, wherein the target nucleic acid comprises a PAM sequence that is compatible with the Cas effector complex.

66. The system of claim 65, wherein the PAM sequence comprises SEQ ID NO:

31.

67. The system of any one of claims 65 to 66, wherein the PAM sequence is located about 50 to about 70 base pairs from the target nucleic acid site.

68. The system of claim 67, wherein the PAM sequence is located 3' to the target nucleic acid site.

69. The system of claim 67, wherein the PAM sequence is located 5' of the target nucleic acid site.

70. The system of any one of claims 62 to 69, wherein the class 2 type V Cas effector is a Cas12k effector.

71. The system of any one of claims 62 to 69, wherein the Class 2 type V Cas effector comprises a polypeptide comprising a sequence at least 80% identical to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220.

72. The system of any one of claims 62 to 69, wherein the Class 2 type V Cas effector comprises a polypeptide comprising a sequence at least 90% identical to any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220.

73. The system of any one of claims 62 to 69, wherein the Class 2 type V Cas effector comprises a polypeptide comprising the sequence of any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85, and 220.

74. The system of any one of claims 62 to 73, wherein the TnsB subunit comprises a polypeptide having a sequence at least 80% identical to any one of SEQ ID NOs: 2, 13, 17 and 65.

75. The system of any one of claims 62 to 73, wherein the TnsB subunit comprises a polypeptide having a sequence at least 90% identical to any one of SEQ ID NOs: 2, 13, 17 and 65.

76. The system of any one of claims 62 to 73, wherein the TnsB subunit comprises a polypeptide having a sequence of any one of SEQ ID NOs: 2, 13, 17 and 65.

77. The system of any one of claims 62 to 76, wherein the Tn7-type 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 to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111.

78. The system of any one of claims 62 to 76, wherein the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide each independently comprising a sequence that is at least 90% identical to any one of SEQ ID NOs: 3-4, 14-15, 18-19, 66-67, and 109-111.

79. The system of any one of claims 62 to 76, wherein the Tn7-type 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 NOs: 3-4, 14-15, 18-19, 66-67, and 109-111.

80. The system of any one of claims 62 to 79, wherein the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least 80% identity to any one of SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222.

81. The system of any one of claims 62 to 79, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 106, 107, 108, 5, 45-63, 68-75, 96-103, and 123-140.

82. The system of any one of claims 62 to 81, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 187-189.

83. The system of any one of claims 62 to 81, wherein the small prokaryotic ribosomal protein subunit S15 is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 181-183.

84. The system of any one of claims 62 to 84, wherein the class 2 type V Cas effector and the Tn7-type transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

85. An engineered nuclease system comprising: a) an endonuclease comprising a RuvC domain, the endonuclease being derived from an uncultured microorganism and being a class 2 VK-type Cas effector, the class 2 VK-type Cas effector comprising at least 80% identity with any one of SEQ ID NOs: 1, 12, 16, 20-30, 64, 80-85 and 220; and b) an engineered guide RNA configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence.

86. The engineered nuclease system of claim 85, wherein the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 5-6, 32-33, 94-95, 104-105, 119-122, and 222.

87. The engineered nuclease system of claim 85 or 86, wherein the engineered guide polynucleotide comprises a sequence at least 80% identical to any one of SEQ ID NOs: 106, 107, 108, 5, 45-63, 68-75, 96-103, and 123-140.

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

89. A cell comprising the system according to any one of claims 1 to 87.

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

91. The cell of claim 89, wherein the cell is a mammalian cell.

92. The cell of claim 89, wherein the cell is an immortalized cell.

93. The cell of claim 89, wherein the cell is an insect cell.

94. The cell of claim 89, wherein the cell is a yeast cell.

95. The cell of claim 89, wherein the cell is a plant cell.

96. The cell of claim 89, wherein the cell is a fungal cell.

97. The cell of claim 89, wherein the cell is a prokaryotic cell.

98. The cell of claim 89, 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 cell, HT1080, HepG2, Huh7, K562, a primary cell, or a derivative thereof.

99. The cell of claim 89, wherein the cell is an engineered cell.

100. The cell of claim 89, wherein the cell is a stable cell.

Images