Fusion protein

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 or transposase 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 or transposase complex, wherein the recombinase or transposase complex is configured to recruit cargo nucleotides 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 / 313,183, filed on February 23, 2022, and U.S. Provisional Patent Application No. 63 / 375,352, filed on September 12, 2022, each of which is incorporated herein in its entirety.

[0003] Sequence Listing

[0004] The contents of the electronic sequence listing (MTG-012WO_SL.xml; size: 524,535 bytes; and creation date: February 23, 2023) are incorporated herein by reference in its entirety. Background Art

[0005] Cas enzymes and their associated clustered regularly interspaced short palindromic repeats (CRISPR) guide RNAs appear to be universal (~45% of bacteria, ~84% of archaea) components of the prokaryotic immune system, 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 fusion protein comprising: (a) 2 types of V-type Cas effectors; and (b) a functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD). In some embodiments, the functional domain is derived from the central globular domain of human histone 1, HMGN1 or Saccharolobus solfataricus sso7d. In some embodiments, the Cas effector is derived from a CAST locus. In some embodiments, the Cas effector comprises a sequence having at least 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% sequence identity with the Cas domain of any one of SEQ ID NOs: 267-268 or a variant thereof. In some embodiments, the functional domain comprises a sequence having at least 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% sequence identity to any one of SEQ ID NOs: 264-266 or variants thereof. In some embodiments, the fusion protein comprises a sequence having at least 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% sequence identity to any one of SEQ ID NOs: 267-268 or variants thereof.

[0007] In some aspects, the present disclosure provides a fusion protein comprising: (a) a TniQ protein; and (b) a functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD). In some embodiments, the TniQ protein is derived from a CAST locus. In some embodiments, the TniQ protein comprises a sequence having at least 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% sequence identity with the TniQ domain of SEQ ID NO: 270 or a variant thereof. In some embodiments, the functional domain comprises a sequence having at least 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% sequence identity to any one of SEQ ID NOs: 264-266 or variants thereof. In some embodiments, the fusion protein comprises a sequence having at least 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% sequence identity to SEQ ID NO: 270 or variants thereof.

[0008] 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 the cargo nucleotide sequence, wherein the cargo nucleotide sequence is configured to interact with a recombinase or transposase complex; a Cas effector complex, the Cas effector complex comprising 2 types of type II Cas effectors and at least one engineered guide polynucleotide configured to hybridize with the target nucleic acid site; and the recombinase or transposase complex, wherein the recombinase or transposase complex is configured to recruit the cargo nucleotide sequence to the target nucleic acid site. In some embodiments, the recombinase or transposase complex is non-covalently bound to the Cas effector complex. In some embodiments, the recombinase or transposase complex is covalently linked to the Cas effector complex. In some embodiments, the recombinase or transposase complex is fused to the Cas effector complex in a single polypeptide. 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 3' to the target nucleic acid site. In some embodiments, the recombinase or transposase complex is a Tn7-type transposase complex. In some embodiments, the engineered guide polynucleotide is configured to bind to the 2 class II type Cas effectors. In some embodiments, the 2 class II type Cas effectors include a polypeptide having a sequence with at least 80% identity to SEQ ID NO: 1 or a variant thereof. In some embodiments, the recombinase or transposase complex comprises at least one, at least two, at least three or four polypeptides, the polypeptide comprising a sequence with at least 80% identity to any one of SEQ ID NO: 2-5 or a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides having at least 80% identity to SEQ ID NO: 12 or a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence with at least 80% identity to SEQ ID NO: 11 or a variant thereof. In some embodiments, the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 17-18 or variants thereof. In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 19 or variants thereof. In some embodiments, the 2 class II type Cas effectors and the recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0009] 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.

[0010] 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 2 types of V-type Cas effectors and an engineered guide polynucleotide 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 TnsA subunit. 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 2 types of V-type Cas effectors are not 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 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 5' of the target nucleic acid site. In some embodiments, the engineered guide polynucleotide is configured to bind to the 2-type V-type Cas effector. In some embodiments, the TnsA subunit comprises a polypeptide having a sequence with at least 80% identity to SEQ ID NO:7 or a variant thereof. In some embodiments, the Tn7-type transposase complex comprises at least one, at least two or three polypeptides, the polypeptide comprising a sequence with at least 80% identity to any one of SEQ ID NO:8-10 or a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence of at least about 46-80 consecutive nucleotides comprising at least 80% identity to any one of SEQ ID NO:13-16 or a variant thereof. In some embodiments, the left recombinase sequence comprises a sequence with at least 80% identity to SEQ ID NO:20 or a variant thereof. In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 21 or a variant thereof. In some embodiments, the Class 2 Type V Cas effector is not a Cas12k effector. 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.

[0011] 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.

[0012] 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 a cargo nucleotide sequence with: a Cas effector complex comprising 2 class II type Cas effectors and at least one engineered guide polynucleotide configured to hybridize with the target nucleic acid site; a recombinase or transposase complex configured to recruit the cargo nucleotide to the target nucleic acid site; and a second double-stranded nucleic acid comprising the target nucleic acid site. In some embodiments, the recombinase or transposase complex binds to the Cas effector complex non-covalently. In some embodiments, the recombinase or transposase complex is covalently linked to the Cas effector complex. In some embodiments, the recombinase or transposase complex is fused to the Cas effector complex in a single polypeptide. 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 target nucleic acid 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 recombinase or transposase complex is a Tn7-type transposase complex. In some embodiments, the engineered guide polynucleotide is configured to bind to the 2nd type II Cas effector. In some embodiments, the 2nd type II Cas effector comprises a polypeptide having a sequence with at least 80% identity to SEQ ID NO: 1 or a variant thereof. In some embodiments, the recombinase or transposase complex comprises at least one, at least two, at least three or four polypeptides, the polypeptide comprising a sequence with at least 80% identity to any one of SEQ ID NO: 2-5 or a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides with at least 80% identity to SEQ ID NO: 12 or a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence with at least 80% identity to SEQ ID NO: 11 or a variant thereof. In some embodiments, the left recombinase sequence comprises a sequence with at least 80% identity to any one of SEQ ID NO: 17-18 or a variant thereof. In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 19 or a variant thereof. In some embodiments, the 2 class II type Cas effectors and the Tn7 type transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0013] 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 a 2-type V-type Cas effector and at least one engineered guide polynucleotide configured to hybridize with the target nucleotide sequence; a Tn7-type transposase complex configured to bind to the Cas effector complex, wherein the Tn7-type transposase complex comprises a TnsA subunit; and a second double-stranded nucleic acid comprising the target nucleic acid site. In some embodiments, the transposase complex non-covalently binds 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 cargo nucleotide sequence is flanked by a left transposase recognition sequence and a right transposase recognition sequence. In some embodiments, the target nucleic acid site 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 TnsA subunit includes a polypeptide having a sequence with at least 80% identity to SEQ ID NO:7 or a variant thereof. In some embodiments, the Tn7-type transposase complex includes at least one, at least two or three polypeptides, the polypeptide including a sequence with at least 80% identity to any one of SEQ ID NO:8-10 or a variant thereof. In some embodiments, the engineered guide polynucleotide includes a sequence of at least about 46-80 consecutive nucleotides comprising at least 80% identity to any one of SEQ ID NO:13-16 or a variant thereof. In some embodiments, the left recombinase sequence includes a sequence with at least 80% identity to SEQ ID NO:20 or a variant thereof. In some embodiments, the right recombinase sequence includes a sequence with at least 80% identity to SEQ ID NO:21 or a variant thereof. In some embodiments, the 2 types of V-type Cas effectors are not Cas12k effectors. 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.

[0014] 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 class I IF-type Cas effector and an engineered guide polynucleotide 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 TnsA subunit. 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 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 3' of the target nucleic acid site. In some embodiments, the PAM sequence is located 5' of the target nucleic acid site. In some embodiments, the engineered guide polynucleotide is configured to bind to the class I IF type Cas effector. In some embodiments, the class I IF type Cas effector includes a polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 41-43 and 48-50 or a variant thereof. In some embodiments, the Tn7 type transposase complex includes at least one, at least two or three polypeptides, the polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 44-46 or 51-53 or a variant thereof.

[0015] In some aspects, the present 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 of the aspects or embodiments described herein in a cell or introducing the system of any of the aspects or embodiments described herein into a cell.

[0016] 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 comprising a cargo nucleotide sequence configured to interact with a Tn7-type transposase complex; a Cas effector complex comprising 2 type V Cas effectors and an engineered guide polynucleotide configured to hybridize with the target nucleotide sequence; and a Tn7-type transposase complex configured to bind to the Cas effector complex, wherein the Tn7-type transposase complex comprises TnsB, TnsC, and TniQ components, wherein: (a) the 2 type V Cas effectors comprise a polypeptide having a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147, or a variant thereof; or (b) the Tn7-type transposase complex comprises a TnsB, TnsC, or TniQ component having a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147, or a variant thereof; ID NO: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107 and 148-150 or a variant thereof having a sequence with at least 80% sequence identity. 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 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NO: 22, 26, 30, 34, 55-89, 104 and 147 or a variant thereof. In some embodiments, the Tn7-type transposase complex includes TnsB, TnsC or TniQ components, and the TnsB, TnsC or TniQ components include sequences with at least 80% sequence identity to any one of SEQ ID NO: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107 and 148-150 or variants 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' of the target nucleic acid site.In some embodiments, the PAM sequence comprises 5'-nGTn-3' or 5'-nGTt-3'. In some embodiments, the engineered guide polynucleotide is configured to bind to the class 2 type V Cas effector. In some embodiments, the TnsB, TnsC or TniQ components respectively comprise a polypeptide having a sequence having at least 80% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107 and 148-150. 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: 90, 91, 92, 93, 117, 151, 156-181 and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 111-114 or 201-206, 255, 262, 256, 209, 257, 263, 258, 210, or a variant thereof. In some embodiments, the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134, or a variant thereof. In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155, 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. In some embodiments: (a) the Class 2 V-type Cas effector comprises a sequence having at least 80% sequence identity with SEQ ID NO: 22 or a variant thereof; (b) the left recombinase sequence comprises a sequence having at least 80% sequence identity with SEQ ID NO: 125 or a variant thereof; (c) the right recombinase sequence comprises a sequence having at least 80% identity with SEQ ID NO: 126 or 155 or a variant thereof; (d) the engineered guide polynucleotide: (i) comprises a sequence having at least 80% sequence identity with at least about 46-60 nucleotides of SEQ ID NO: 90; or (ii) comprises a sequence having at least 80% sequence identity with non-degenerate nucleotides of any one of SEQ ID NO: 94, 112 or 202; or (e) the TnsB, TnsC and TniQ components comprise sequences having at least 80% sequence identity with SEQ ID NO: 23-25 ​​or a variant thereof.In some embodiments: (a) the Class 2 V-type Cas effector comprises a sequence having at least 80% sequence identity to SEQ ID NO: 26 or a variant thereof; (b) the left recombinase sequence comprises a sequence having at least 80% sequence identity to SEQ ID NO: 127 or a variant thereof; (c) the right recombinase sequence comprises a sequence having at least 880% sequence identity to SEQ ID NO: 128 or a variant thereof; (d) the engineered guide polynucleotide: (i) comprises a sequence having at least 80% sequence identity to at least about 46-60 nucleotides of any one of SEQ ID NO: 91, 156 or 209; or (ii) comprises a sequence having at least 80% sequence identity to non-degenerate nucleotides of any one of SEQ ID NO: 95, 113 or 203; or (e) the TnsB, TnsC and TniQ components comprise sequences having at least 80% sequence identity to SEQ ID NO: 27-29 or variants 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: 60 or a variant thereof; (b) the left recombinase sequence comprises a sequence having at least 80% sequence identity with SEQ ID NO: 131 or a variant thereof; (c) the right recombinase sequence comprises a sequence having at least 80% sequence identity with SEQ ID NO: 132 or a variant thereof; (d) the engineered guide polynucleotide: (i) comprises a sequence having at least 80% sequence identity with at least about 46-60 nucleotides of any one of SEQ ID NO: 117, 161 or 214; or (ii) comprises a sequence having at least 80% sequence identity with non-degenerate nucleotides of SEQ ID NO: 119; or (e) the TnsB, TnsC and TniQ components comprise sequences having at least 80% sequence identity with SEQ ID NO: 101-103 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: 147 or a variant thereof; (b) the left recombinase sequence comprises a sequence having at least 80% sequence identity with SEQ ID NO: 153 or a variant thereof; (c) the right recombinase sequence comprises a sequence having at least 880% sequence identity with SEQ ID NO: 154 or a variant thereof; (d) the engineered guide polynucleotide: (i) comprises a sequence having at least 80% sequence identity with at least about 46-60 nucleotides of any one of SEQ ID NO: 151, 181 or 234; or (ii) comprises a sequence having at least 80% sequence identity with non-degenerate nucleotides of SEQ ID NO: 152 or 254; or (e) the TnsB, TnsC and TniQ components comprise sequences having at least 80% sequence identity with SEQ ID NO: 148-150 or a variant thereof. In some embodiments: (a) the Class 2 V-type Cas effector comprises a sequence having at least 80% sequence identity to SEQ ID NO: 34 or a variant thereof; (b) the left recombinase sequence comprises a sequence having at least 80% sequence identity to SEQ ID NO: 129 or a variant thereof; (c) the right recombinase sequence comprises a sequence having at least 880% sequence identity to SEQ ID NO: 130 or a variant thereof; (d) the engineered guide polynucleotide: (i) comprises a sequence having at least 80% sequence identity to at least about 46-60 nucleotides of any one of SEQ ID NO: 93, 157 or 210; or (ii) comprises a sequence having at least 80% sequence identity to non-degenerate nucleotides of any one of SEQ ID NO: 97, 114 or 204; or (e) the TnsB, TnsC and TniQ components comprise sequences having at least 80% sequence identity to SEQ ID NO: 148-150 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: 30 or a variant thereof; (b) the left recombinase sequence comprises a sequence having at least 80% sequence identity with SEQ ID NO: 123 or a variant thereof; (c) the right recombinase sequence comprises a sequence having at least 80% identity with SEQ ID NO: 124 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: 92; or (ii) comprises a sequence having at least 80% identity with non-degenerate nucleotides of SEQ ID NO: 111 or 201; (e) the TnsB, TnsC and TniQ components comprise polypeptides having sequences having at least 80% identity with SEQ ID NO: 31, 32 and 33 or variants thereof; or (f) the PAM sequence comprises 5'-nGTn-3' or 5'-nGTt-3'.

[0017] 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 comprising a cargo nucleotide sequence configured to interact with a Tn7-type transposase complex; a Cas effector complex comprising 2 types of V-type Cas effectors and an engineered guide polynucleotide 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 and TnsC components but does not include TnsA and / or 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 Tn7-type transposase complex comprises a sequenced polypeptide having at least 80% sequence identity to any one of SEQ ID NOs: 39-40 and 109-110. In some embodiments, the TnsB component comprises a polypeptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 40 or 109. In some embodiments, the TnsC component comprises a polypeptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 39 or 110. In some embodiments, the 2nd type V-type Cas effector is a Cas12k effector. In some embodiments, the 2nd type V-type Cas effector comprises a sequence having at least 80% sequence identity to SEQ ID NO: 38 or SEQ ID NO: 108. 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 double-stranded nucleic acid or the system comprising the target nucleic acid site is within a cell. 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 engineered guide polynucleotide is configured to bind to the Class 2 Type V Cas effector. In some embodiments, the TnsB and TnsC components comprise a polypeptide having a sequence having at least 80% identity to SEQ ID NOs: 40 and 39 or 109 and 110, respectively.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: 118, 182, 183, 235, and 236, or variants thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% identity to non-degenerate nucleotides of any one of SEQ ID NOs: 115, 116, 205, 206, 261, 235, 260, and 236, or variants thereof. In some embodiments, the left recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 134. In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 135, or variants 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. In some embodiments, (a) the Class 2 V-type Cas effector comprises a sequence having at least 80% sequence identity with SEQ ID NO:38 or a variant thereof; (b) the left recombinase sequence comprises a sequence having at least 80% sequence identity with SEQ ID NO:134 or a variant thereof; (c) the right recombinase sequence comprises a sequence having at least 80% identity with SEQ ID NO:135 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:182 or 235; or (ii) comprises a sequence having at least 80% identity with non-degenerate nucleotides of SEQ ID NO:98, 115, 116, 205 and 206; or (e) the TnsB and TnsC components comprise polypeptides having sequences having at least 80% identity with SEQ ID NO:40 and 39 or variants thereof.

[0018] In some aspects, the present disclosure provides an engineered nuclease system, the system comprising: an endonuclease comprising a RuvC domain and a HNH domain, wherein the endonuclease is derived from an uncultured microorganism, wherein the endonuclease is a Class 2 type II endonuclease comprising a sequence having at least 80% identity with SEQ ID NO: 1 or a variant thereof; and an engineered guide polynucleotide, wherein the engineered guide polynucleotide is configured to form a complex with the endonuclease, and the engineered guide polynucleotide comprises a spacer sequence configured to hybridize with a target nucleic acid sequence. In some embodiments, the engineered guide polynucleotide comprises at least 60-80 consecutive nucleotides having at least 80% identity with SEQ ID NO: 12 or a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% identity with SEQ ID NO: 11 or a variant thereof.

[0019] In some aspects, the present disclosure provides an engineered nuclease system, the 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 V-type endonuclease having at least 80% identity to SEQ ID NO: 5; and an engineered guide polynucleotide, wherein the engineered guide polynucleotide 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 SEQ ID NO: 13-16 or a variant thereof.

[0020] In some aspects, the present disclosure provides an engineered nuclease system, the 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 endonuclease having at least 80% identity with any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147, or a variant thereof; and an engineered guide polynucleotide, wherein the engineered guide polynucleotide is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a spacer sequence configured to hybridize with 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 with any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234, or a variant 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: 111-114 or 201-206, 255, 262, 256, 209, 257, 263, 258, 210, or variants thereof.

[0021] In some aspects, the present disclosure provides an engineered nuclease system, the 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 endonuclease having at least 80% identity with any one of SEQ ID NO: 38 or SEQ ID NO: 108 or a variant thereof; and an engineered guide polynucleotide, wherein the engineered guide polynucleotide is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a spacer sequence configured to hybridize with 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 with any one of SEQ ID NO: 118, 182, 183, 235 and 236 or a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least 80% identical to non-degenerate nucleotides of any one of SEQ ID NOs: 111-114 or 201-206, 255, 262, 256, 209, 257, 263, 258, 210, 115, 116, 205, 206, 261, 235, 260, or 236, or variants thereof.

[0022] In some aspects, the present disclosure provides an engineered nuclease system, the system comprising: a Class I IF-type Cas endonuclease, the Class I IF-type Cas endonuclease comprising at least one Cas6, Cas7 or Cas8 polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 41-43 and 48-50 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 having at least 80% identity to a non-degenerate nucleotide of any one of SEQ ID NOs: 121, 122, 207 and 208.

[0023] 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 2 class II type Cas effectors, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide configured to hybridize with the target nucleic acid site; a recombinase or transposase complex, the recombinase or transposase complex being configured to bind to the Cas effector complex; a double-stranded nucleic acid, the double-stranded nucleic acid being configured to interact with the recombinase or transposase complex and comprising the cargo nucleotide sequence; and a functional domain, the functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0024] In some embodiments, the Cas effector complex is non-covalently bound to the recombinase or transposase complex. In some embodiments, the Cas effector complex is covalently linked to the recombinase or transposase complex. In some embodiments, the Cas effector complex is fused to the recombinase or transposase complex.

[0025] In some embodiments, the cargo nucleotide sequence is flanked by a left transposase recognition sequence and a right transposase recognition sequence recognized by the recombinase or transposase complex. In some embodiments, the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 17-18. In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 19.

[0026] In some embodiments, the system further comprises a PAM sequence compatible with the Cas effector complex. 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.

[0027] In some embodiments, the 2 class II type Cas effectors include a polypeptide comprising a sequence having at least 80% identity to SEQ ID NO: 1. In some embodiments, the 2 class II type Cas effectors include a polypeptide comprising a sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, the 2 class II type Cas effectors include a polypeptide comprising a sequence of SEQ ID NO: 1.

[0028] In some embodiments, the recombinase or transposase complex is a Tn7-type transposase complex. In some embodiments, the recombinase or transposase complex comprises at least one polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 2-5. In some embodiments, the recombinase or transposase complex comprises at least one polypeptide comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 2-5. In some embodiments, the recombinase or transposase complex comprises at least one polypeptide comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 2-5.

[0029] In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least 80% identity to SEQ ID NO: 12. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to SEQ ID NO:11.

[0030] In some embodiments, the functional domain is derived from human histone 1 central globular domain, HMGN1, cbx5, or Sulfolobus solfataricus sso7d. In some embodiments, the functional domain comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 264-266. In some embodiments, the class 2 type II Cas effector is fused to the functional domain to form a fusion protein.

[0031] In some embodiments, the recombinase or transposase complex comprises a TniQ protein. In some embodiments, the TniQ protein is fused to the functional domain to form a fusion protein. In some embodiments, the TniQ protein comprises a sequence having at least 80% sequence identity with the TniQ domain of SEQ ID NO: 270.

[0032] 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: 341-506. 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: 620, 373, 375, 383, 424, 449, 500, and 506.

[0033] In some embodiments, said 2 Class II type Cas effectors and said recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[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 configured to hybridize with the target nucleic acid site; a Tn7-type transposase complex, the Tn7-type transposase complex being configured to bind to the Cas effector complex and comprising TnsA, TnsB, TnsC, and TniQ components; a double-stranded nucleic acid, the double-stranded nucleic acid being configured to interact with the Tn7-type transposase complex and comprising the cargo nucleotide sequence; and a functional domain, the functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0035] 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.

[0036] In some embodiments, the cargo nucleotide sequence is flanked by a left transposase recognition sequence and a right transposase recognition sequence recognized by the recombinase or transposase complex. In some embodiments, the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NO: 20. In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 21.

[0037] In some embodiments, the system further comprises a PAM sequence compatible with the Cas effector complex. 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.

[0038] In some embodiments, the Class 2 type V Cas effector is not a Cas12k effector.

[0039] In some embodiments, the TnsA component comprises a polypeptide comprising a sequence at least 80% identical to SEQ ID NO: 7. In some embodiments, the Tn7-type transposase complex comprises at least one polypeptide comprising a sequence at least 80% identical to any one of SEQ ID NOs: 8-10.

[0040] 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: 13-16.

[0041] In some embodiments, the functional domain is derived from human histone 1 central globular domain, HMGN1, cbx5, or Sulfolobus solfataricus sso7d. In some embodiments, the functional domain comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 264-266. In some embodiments, the class 2 type V Cas effector is fused to the functional domain to form a fusion protein. In some embodiments, the fusion protein comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 267-268.

[0042] In some embodiments, the Tn7 transposase complex comprises a TniQ protein. In some embodiments, the TniQ protein is fused to the functional domain to form a fusion protein. In some embodiments, the TniQ protein comprises a sequence having at least 80% sequence identity with the TniQ domain of SEQ ID NO: 270.

[0043] 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: 341-506. 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: 620, 373, 375, 383, 424, 449, 500, and 506.

[0044] In some embodiments, said 2 Class II type Cas effectors and said recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0045] 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 I IF-type Cas effector, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide configured to hybridize with the target nucleic acid site; a Tn7-type transposase complex, the Tn7-type transposase complex being configured to bind to the Cas effector complex and comprising TnsA, TnsB, TnsC, and TniQ components; a double-stranded nucleic acid, the double-stranded nucleic acid being configured to interact with the Tn7-type transposase complex and comprising a cargo nucleotide sequence; and a functional domain, the functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0046] 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.

[0047] In some embodiments, wherein the cargo nucleotide sequence is flanked by a left transposase recognition sequence and a right transposase recognition sequence recognized by the recombinase or transposase complex. In some embodiments, the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 136 and 138. In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NOs: 137 and 139.

[0048] In some embodiments, the system further comprises a PAM sequence compatible with the Cas effector complex. 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.

[0049] In some embodiments, the class I IF type Cas effector comprises a polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some embodiments, the class I IF type Cas effector comprises a polypeptide comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some embodiments, the class I IF type Cas effector comprises a polypeptide comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 41-43 and 48-50.

[0050] In some embodiments, the Tn7-type transposase complex comprises at least one polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some embodiments, the Tn7-type transposase complex comprises at least one polypeptide comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some embodiments, the Tn7-type transposase complex comprises at least one polypeptide comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 44-47 and 51-54.

[0051] In some embodiments, the functional domain is derived from human histone 1 central globular domain, HMGN1, cbx5, or Sulfolobus solfataricus sso7d. In some embodiments, the functional domain comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 264-266. In some embodiments, the class I IF-type Cas effector is fused to the functional domain to form a fusion protein. In some embodiments, the fusion protein comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 267-268.

[0052] In some embodiments, the Tn7 transposase complex comprises a TniQ protein. In some embodiments, the TniQ protein is fused to the functional domain to form a fusion protein.

[0053] In some embodiments, the TniQ protein comprises a sequence having at least 80% sequence identity to the TniQ domain of SEQ ID NO:270.

[0054] 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: 341-506. 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: 620, 373, 375, 383, 424, 449, 500, and 506.

[0055] In some embodiments, the class I IF-type Cas effector and the recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0056] 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 configured to hybridize to the target nucleic acid site and comprising: i) a Class 2 type V Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147; and ii) an engineered guide polynucleotide comprising a polypeptide having a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147; NO:90-93, 111-114, 117, 151, 156-181, 201-206, 255, 262, 256, 209, 257, 263, 258 and 210 have a sequence with at least 80% sequence identity; 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 include a sequence with at least 80% sequence identity to SEQ ID NO: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107 and 148-150 have at least 80% identity sequence; double-stranded nucleic acid, the double-stranded nucleic acid is configured to interact with the Tn7-type transposase complex and comprises, in 5' to 3' order: i) a left recombinase sequence, the left recombinase sequence comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 125, 127, 123, 129, 131, 133, 153 and 134; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence, the right recombinase sequence comprising a sequence having at least 80% sequence identity with SEQ ID NO: Any one of NO: 124, 126, 128, 130, 132, 154 and 155 has a sequence with at least 80% identity; and a functional domain, wherein the functional domain includes a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0057] 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 configured to hybridize to the target nucleic acid site and comprising: i) a Class 2 V-type Cas effector, the Class 2 V-type Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 22; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 90, 112, and 202; 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 polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 22; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 90, 112, and 202; and NO:23-25 ​​has a sequence with at least 80% identity; 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, the left recombinase sequence comprising a sequence with at least 80% sequence identity to SEQ ID NO: 125; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence, the right recombinase sequence comprising a sequence with at least 80% identity to any one of SEQ ID NO: 126 and 155; and a functional domain, the functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0058] 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 configured to hybridize to the target nucleic acid site and comprising: i) a Class 2 type V Cas effector, the Class 2 type V Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 26; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 91, 113, 156, 203, and 209; 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 polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 26; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 91, 113, 156, 203, and 209; NO:27-29 has a sequence with at least 80% identity; 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, the left recombinase sequence comprising a sequence with at least 80% sequence identity to SEQ ID NO: 127; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence, the right recombinase sequence comprising a sequence with at least 80% identity to SEQ ID NO: 128; and a functional domain, the functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0059] 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 configured to hybridize to the target nucleic acid site and comprising: i) a Class 2 V-type Cas effector, the Class 2 V-type Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 60; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 117, 119, 161, and 214; 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 SEQ ID NO: 117, 119, 161, and 214; NO:101-103 has a sequence with at least 80% identity; 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, the left recombinase sequence comprising a sequence with at least 80% sequence identity to SEQ ID NO:131; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence, the right recombinase sequence comprising a sequence with at least 80% identity to SEQ ID NO:132; and a functional domain, the functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0060] 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 configured to hybridize to the target nucleic acid site and comprising: i) a Class 2 V-type Cas effector, the Class 2 V-type Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 147; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 151, 152, 181, 234, and 254; 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 polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 147; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 151, 152, 181, 234, and 254; NO:148-150 has a sequence with at least 80% identity; 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, the left recombinase sequence comprising a sequence with at least 80% sequence identity to SEQ ID NO:153; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence, the right recombinase sequence comprising a sequence with at least 80% identity to SEQ ID NO:154; and a functional domain, the functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0061] 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 configured to hybridize to the target nucleic acid site and comprising: i) a Class 2 V-type Cas effector, the Class 2 V-type Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 34; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 93, 114, 157, 204, and 210; 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 polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 34; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 93, 114, 157, 204, and 210; NO:148-150 has a sequence with at least 80% identity; 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, the left recombinase sequence comprising a sequence with at least 80% sequence identity to SEQ ID NO:129; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence, the right recombinase sequence comprising a sequence with at least 80% identity to SEQ ID NO:130; and a functional domain, the functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0062] 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 configured to hybridize to the target nucleic acid site and comprising: i) a Class 2 V-type Cas effector, the Class 2 V-type Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 30; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 92, 111, and 201; 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 SEQ ID NO: 30; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 92, 111, and 201; and 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 SEQ ID NO: NO:31-33 has a sequence with at least 80% identity; 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, the left recombinase sequence comprising a sequence with at least 80% sequence identity to SEQ ID NO:123; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence, the right recombinase sequence comprising a sequence with at least 80% identity to SEQ ID NO:124; and a functional domain, the functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0063] 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 configured to hybridize to the target nucleic acid site and comprising: i) a Class 2 V-type Cas effector, the Class 2 V-type Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 38; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 98, 115-116, 182, 205-206, and 235; 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 SEQ ID NO: NO:39 and 40 have a sequence with at least 80% identity; a double-stranded nucleic acid, which is configured to interact with the Tn7-type transposase complex and includes, in 5' to 3' order: i) a left recombinase sequence, the left recombinase sequence includes a sequence with at least 80% sequence identity to SEQ ID NO: 134; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence, the right recombinase sequence includes a sequence with at least 80% identity to SEQ ID NO: 135; and a functional domain, the functional domain including a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0064] In some embodiments, the class 2 type V Cas effector is a Cas12k effector.

[0065] In some embodiments, the system further comprises a PAM sequence 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 5'-nGTn-3' or 5'-nGTt-3'.

[0066] 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: 341-506. 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: 620, 373, 375, 383, 424, 449, 500, and 506.

[0067] 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.

[0068] 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 configured to hybridize with the target nucleic acid site; a Tn7-type transposase complex, the Tn7-type transposase complex being configured to bind to the Cas effector complex and comprising TnsB and TnsC components but not TnsA and / or TniQ components; a double-stranded nucleic acid, the double-stranded nucleic acid being configured to interact with the Tn7-type transposase complex and comprising the cargo nucleotide sequence; and a functional domain, the functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0069] 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.

[0070] In some embodiments, the cargo nucleotide sequence is flanked by a left transposase recognition sequence and a right transposase recognition sequence recognized by the recombinase or transposase complex. In some embodiments, the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NO: 134.

[0071] In some embodiments, the right recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 135. In some embodiments, the system further comprises a PAM sequence compatible with the Cas effector complex. 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' to the target nucleic acid site. In some embodiments, the PAM sequence is located 5' to the target nucleic acid site.

[0072] 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: 38 and 108. 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: 38 and 108. 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: 38 and 108.

[0073] In some embodiments, the TnsB subunit comprises a polypeptide comprising a sequence at least 80% identical to SEQ ID NO:40 or 109. In some embodiments, the TnsC subunit comprises a polypeptide comprising a sequence at least 80% identical to SEQ ID NO:39 or 110.

[0074] In some embodiments, the Tn7-type transposase complex comprises at least one polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 115, 116, 205, 206, 261, 235, 260, and 236.

[0075] 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: 118, 182, 183, 235, and 236.

[0076] 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: 341-506. 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: 620, 373, 375, 383, 424, 449, 500, and 506.

[0077] In some embodiments, said 2 Class II type Cas effectors and said recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0078] In some embodiments, 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 2 class II type Cas effectors, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide, the engineered guide polynucleotide being configured to hybridize with the target nucleic acid; a recombinase or transposase complex, the recombinase or transposase complex being configured to bind to the Cas effector complex; a double-stranded nucleic acid, the double-stranded nucleic acid comprising, in 5' to 3' order: i) a left recombinase recognition sequence; ii) the cargo nucleotide sequence; and iii) a right recombinase recognition sequence, the left recombinase recognition sequence and the right recombinase recognition sequence being capable of being recognized by the recombinase or transposase complex; and a functional domain, the functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0079] In some embodiments, 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 being capable of hybridizing with the target nucleic acid; a Tn7-type transposase complex, the Tn7-type transposase complex being configured to bind to the Cas effector complex and comprising TnsA, TnsB, TnsC, and TniQ components; a double-stranded nucleic acid, the double-stranded nucleic acid comprising, in 5' to 3' order: i) a left recombinase recognition sequence; ii) the cargo nucleotide sequence; and iii) a right recombinase recognition sequence, the left recombinase recognition sequence and the right recombinase recognition sequence being capable of being recognized by the Tn7-type transposase complex; and a functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0080] In some embodiments, 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 I IF-type Cas effector, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide, the engineered guide polynucleotide being capable of hybridizing with the target nucleic acid; a Tn7-type transposase complex, the Tn7-type transposase complex being configured to bind to the Cas effector complex and comprising TnsA, TnsB, TnsC, and TniQ components; a double-stranded nucleic acid, the double-stranded nucleic acid comprising, in 5' to 3' order: i) a left recombinase recognition sequence; ii) the cargo nucleotide sequence; and iii) a right recombinase recognition sequence, the left recombinase recognition sequence and the right recombinase recognition sequence being capable of being recognized by the Tn7-type transposase complex; and a functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0081] In some embodiments, 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 being capable of hybridizing with the target nucleic acid; a Tn7-type transposase complex, the Tn7-type transposase complex being configured to bind to the Cas effector complex and comprising TnsB and TnsC components but not TnsA and / or TniQ components; a double-stranded nucleic acid, the double-stranded nucleic acid comprising, in 5' to 3' order: i) a left recombinase recognition sequence; ii) the cargo nucleotide sequence; and iii) a right recombinase recognition sequence, the left recombinase recognition sequence and the right recombinase recognition sequence being capable of being recognized by the Tn7-type transposase complex; and a functional domain comprising a DNA binding domain (DBD) or a chromatin regulatory domain (CMD).

[0082] In some aspects, the present disclosure provides an engineered nuclease system, the system comprising: a nuclease comprising a RuvC domain and a HNH domain, wherein the nuclease is derived from an uncultured microorganism, wherein the nuclease is a Class 2, Type II endonuclease comprising a sequence having at least 80% identity with SEQ ID NO: 1; and an engineered guide polynucleotide, wherein the engineered guide RNA is configured to form a complex with the nuclease, and the engineered guide RNA comprises a spacer sequence configured to hybridize to a target nucleic acid sequence.

[0083] In some embodiments, the engineered guide polynucleotide comprises at least 60-80 contiguous nucleotides having at least 80% identity to SEQ ID NO:12.

[0084] In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least 80% identical to SEQ ID NO:11.

[0085] An engineered nuclease system, the 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 V-type endonuclease having at least 80% identity to SEQ ID NO:5; 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.

[0086] 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: 13-16.

[0087] An engineered nuclease system, the system comprising: a nuclease comprising a RuvC domain, wherein the nuclease is derived from an uncultured microorganism, and wherein the nuclease is a Class 2 VK-type nuclease having at least 80% identity with any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147; and an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the nuclease, and the engineered guide RNA comprises a spacer sequence configured to hybridize to a target nucleic acid sequence.

[0088] 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: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 111-114, 201-206, 255, 262, 256, 209, 257, 263, 258, and 210.

[0089] In some aspects, the present disclosure provides an engineered nuclease system, the system comprising: a nuclease comprising a RuvC domain, wherein the nuclease is derived from an uncultured microorganism, and wherein the nuclease is a Class 2 VK-type nuclease having at least 80% identity with SEQ ID NO:38 or SEQ ID NO:108; and an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the nuclease, and the engineered guide RNA comprises a spacer sequence configured to hybridize to a target nucleic acid sequence.

[0090] 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: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 111-114 or 201-206, 255, 262, 256, 209, 257, 263, 258, 210, 115, 116, 205, 206, 261, 235, 260, and 236.

[0091] In some aspects, the present disclosure provides an engineered nuclease system, the system comprising: a Class I IF-type Cas endonuclease, the Class I IF-type Cas endonuclease comprising at least one Cas6, Cas7 or Cas8 polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 41-43 and 48-50; 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 having at least 80% identity to any one of SEQ ID NOs: 121, 122, 207 and 208.

[0092] In some aspects, the present disclosure provides a method for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the method comprising introducing the system of the present disclosure into a cell. In some aspects, the present disclosure provides a cell comprising 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 a derivative thereof. In some embodiments, the cell is an engineered cell. In some embodiments, the cell is a stable cell.

[0093] Other aspects and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described in the detailed description. As will be appreciated, the present disclosure is capable of other and 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

[0094] 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.)"):

[0095] Figure 1 Example organizations of different species and types of CRISPR / Cas loci are depicted.

[0096] Figure 2 Depicted is the architecture of the native 2 type II crRNA / tracrRNA pairs compared to the hybrid sgRNA in which the crRNA and tracrRNA are linked.

[0097] Figure 3 Two pathways found in Tn7 and Tn7-like elements are depicted.

[0098] Figures 4A-4C The genomic context of the type II Tn7-reduced CAST of the MG36 family is depicted. Figure 4A The MG36-5CAST system is shown to include a CRISPR array (CRISPR repeats), a type II nuclease with RuvC and HNH endonuclease domains, and four predicted transposase protein open reading frames. The catalytic transposase TnsB is encoded as two subunits. Figure 4B The two transposon ends predicted for the MG36-1 CAST system (TIR-1 and TIR-2) are shown. Figure 4C An alignment of the predicted left (LE) and right (RE) end sequences of the type II Tn7-reduced CAST transposon is shown, with annotated repeat sequences indicated by arrows. The left and right ends are labeled according to their orientation.

[0099] Figure 5A-5B The genomic context of the V-type Tn7 CAST of the MG39 family is depicted. Figure 5A The MG39-1 CAST system is shown to include a V-type nuclease, four predicted transposon proteins (TnsABC and TniQ), and a CRISPR array. Transposon ends are predicted for the MG39-1 CAST system (TIR-1). Figure 5B An alignment of the predicted left (LE) and right (RE) end sequences of the V-type Tn7 CAST transposon is shown, with annotated inverted repeat sequences indicated by arrows.

[0100] Figure 6 and Figure 7 Depicted are predicted structures for sgRNAs by the CAST system described herein (eg, predicted in Example 3).

[0101] Figure 8 The genomic background of MG108-1 (system described herein) is depicted. This candidate is a Cas12K CAST that naturally lacks TniQ. Genes in the genomic fragment are indicated by arrows.

[0102] Fig. 9 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.

[0103] Figures 10A-10C MG110 Cascade CAST is depicted. Fig. 10AThe genomic context of the MG110-1 Cascade CAST is shown. The complete Tn7 suite (TnsA, TnsB, TnsC / TniB, TniQ) and the defective Cascade suite (Cas6, Cas7, fused Cas5-Cas8) are indicated by orange arrows. The TIRs flanking the CAST transposon are indicated by connected arrows. Fig. 10B The secondary structure of the repeat sequence indicating the crRNA stem-loop structure is depicted. Fig. 10C The sequence alignment of CRISPR repeat sequences from A. wodanis, V. cholerae and MG110 family CAST shown in Figure 4 indicates conserved motifs indicating crRNA stem-loop secondary structure.

[0104] 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.

[0105] 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 position 92-98 (top frame) is considered to be important for sgRNA tertiary structure and non-continuous repeat sequence-anti-repeat sequence pairing with crRNA. The hairpin "CYCC (n6) GGRG" at position 265-278 (bottom frame) may be important for function and may locate the downstream sequence of crRNA pairing.

[0106] Fig. 11C The presence of a repeat-antirepeat (RAR) motif is shown in, for example, the MG64-2, MG64-4, MG64-5, MG64-6, MG64-7, and MG108-1 families.

[0107] Fig. 12A The predicted structure of the MG64-2 sgRNA is depicted.

[0108] Fig. 12B The predicted structure of the MG64-4 sgRNA is depicted.

[0109] Fig. 12C The predicted structure of the MG64-6 sgRNA is depicted.

[0110] Fig.12D The predicted structure of the MG64-7 sgRNA is depicted.

[0111] Fig.12E The predicted structure of the MG108-1 sgRNA is depicted.

[0112] Figures 13A-13C Describe PCR, PAM and Sanger sequencing (Sanger sequencing) data, these data prove that MG64-6 is active in vitro.Use described in vitro targeted integrase activity scheme, express effector protein and its TnsB, TnsC and TniQ protein in in vitro transcription / translation system.After translation, target DNA, cargo DNA and sgRNA are added to reaction buffer.Integrate by PCR assay across target / donor junction. Fig.13A Gel images of transposition PCRs are depicted, showing apo (no sgRNA) and 64-6 (with sgRNA 64-6sgRNA). PCR 3 detects the RE junction distal to the PAM. PCR 4 is the LE junction distal to the PAM. PCR 5 is the RE junction proximal to the PAM. PCR 6 is the LE junction proximal to the PAM. PCRs are paired in different possible orientations (PCR 3 and 6 vs. PCR 4 and 5). LE-PAM proximal and RE-PAM distal orientations are preferred. Fig. 13B PAMs from in vitro transposition assays, sequencing PCRs 5 and 6 are depicted. Fig. 13C Describe Sanger data (Sanger data), described data show the junction point of transposition that excision occurs in donor DNA.First small figure shows PCR 3 and PCR 5 (RE).Second small figure shows PCR 4 and PCR 6 (LE).Sanger sequencing reaction is the reaction of donor-target product, and when junction point occurs, sequencing stops to mate with donor DNA (dark bar below sequencing peak).

[0113] Fig.14 Depicted are next generation sequencing (NGS) results of in vitro transposition products that reveal insertion site preferences. NGS reads were processed in CRISPResso2 and compared to a reference sequence transposed at position 60. The resulting indels correspond to transpositions earlier or later than this arbitrary reference sequence.

[0114] Fig.15Electrophoretic mobility shift assay (EMSA) results of 64-2TnsB and its RE 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 RE 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 lead to 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. The upward shift of the labeled band in lane 3 indicates that TnsB binds to the RE sequence, indicating that the RE sequence contains an active RE transposition sequence.

[0115] Fig.16 Depicted are the activities of Cas12k and TniQ fusions tested in vitro. Fig.16 Panel A depicts a gel image showing transposition activity of the left end to the donor. Lane 1 = apo (without sgRNA), Lane 2 = holo (with sgRNA), Lane 3 = MG64-6-Cas12k-sso7d-NLS, Lane 4 = NLS-sso7d-MG64-6-Cas12k, Lane 5 = MG64-6-Q-H1 Core-NLS, Lane 6 = MG64-6-Q-HMGN1-NLS. Fig.16 Panel B depicts a gel image showing transposition activity of the left end to the donor. Lane 1 = apo (no sgRNA), Lane 2 = holo (with sgRNA), Lane 3 = cytoplasmic MG64-6-Cas12k-sso7d-NLS, Lane 4 = cytoplasmic MG64-6-Q-H1 core-NLS, Lane 5 = skip, Lane 6 = skip, Lane 7 = nuclear MG64-6-Cas12k-sso7d-NLS, Lane 8 = nuclear MG64-6-Q-H1 core-NLS.

[0116] Fig.17 A tree demonstrating that MG161 family members are distant homologs of sso7d is depicted. The tree was inferred from a multiple sequence alignment of full-length protein sequences containing PFamPF02294 domain hits. The reference sso7d sequence is highlighted with triangles. The distance between tips is estimated to be 0.5 substitutions per site (horizontal bars).

[0117] Fig.18A The genomic context of a protein encoding multiple functional domains (FD) is depicted. The FDs correspond to tandem imperfect repeats (arrows labeled 161-12 to 161-18).

[0118] Fig.18BDepicted is a multiple sequence alignment of tandem repeat FDs with a reference sso7d sequence from Sulfolobus solfataricus. MG161-13 has 20% amino acid identity (AAI) with the reference sequence, while the other FDs have lower sequence identities.

[0119] Fig.19 A tree demonstrating that MG162 family members are distant homologs of HMGN1 is depicted. The tree was inferred from a multiple sequence alignment of full-length protein sequences containing PFamPF01101 domain hits. The reference HMGN1 sequence is highlighted with triangles. The distance between the tips is estimated to be 0.3 substitutions per site (horizontal bars).

[0120] Fig. 20 A multiple sequence alignment of the MG162 functional domain protein with reference human and mouse HMGN1 sequences is depicted. The average pairwise percent identity of the alignment is 40.4%. The conserved RXSXRLS motif is highlighted with a black box.

[0121] Fig.21 A schematic diagram depicting the identification of ribosomal protein S15 homologs in Cyanobacteria genomic fragments. Candidate sequences from the same sample from which the Cas12k effector was recovered are highlighted with black dots. Reference S15 from Escherichia coli is shown with an arrow.

[0122] Brief Description of Sequence Listing

[0123] 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.

[0124] MG36

[0125] SEQ ID NO: 1 shows the full-length peptide sequence of the MG36 Cas effector.

[0126] SEQ ID NO: 2-5 shows the peptide sequence of MG36 transposase protein, which may include

[0127] Cas effector-associated recombinase or transposase complex. The addition of -B1, -B2, -T1, and -C at the end of the tag indicates similarity to the TnsB1, TnsB2, TnsT1, and TniC proteins of the Tn7-like system, respectively.

[0128] SEQ ID NO: 11 shows the nucleotide sequence of the sgRNA engineered to function with the MG36 Cas effector.

[0129] SEQ ID NO: 12 shows MG36 derived from the same locus as the MG36 Cas effector

[0130] Nucleotide sequence of tracrRNA.

[0131] SEQ ID NOs: 17-18 show the nucleotide sequences of the left transposase recognition sequences associated with the MG36 system

[0132] SEQ ID NO: 19 shows the nucleotide sequence of the right transposase recognition sequence associated with the MG36 system.

[0133] MG39

[0134] SEQ ID NO:6 shows the full-length peptide sequence of the MG39-1 Cas effector.

[0135] SEQ ID NOs: 7-10 show peptide sequences of MG39-1 transposase proteins, which may include a recombinase or transposase complex associated with an MG39-1 Cas effector.

[0136] SEQ ID NOs: 13-16 show MG39 derived from the same locus as the MG39 Cas effector

[0137] Nucleotide sequence of tracrRNA.

[0138] SEQ ID NO: 20 shows the nucleotide sequence of the left-side transposase recognition sequence associated with the MG39 system.

[0139] SEQ ID NO: 21 shows the nucleotide sequence of the right transposase recognition sequence associated with the MG39 system.

[0140] MG64

[0141] SEQ ID NOs: 22, 26, 30, 34, 55-89, 104 and 147 show the full-length peptide sequences of the MG64 Cas effectors.

[0142] SEQ ID NO: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107 and 148-150 show peptide sequences of MG64 transposase proteins, which may include a recombinase or transposase complex associated with an MG64 Cas effector. -A, -B, -C and -Q are added to the ends of the tags to indicate similarity to the TnsA, TnsB, TnsC and TniQ proteins of the Tn7-like system, respectively.

[0143] SEQ ID NOs: 90-93, 117, 151, 156-181 and 209-234 show the nucleotide sequences of MG64 tracrRNAs derived from the same locus as the MG64 effector.

[0144] SEQ ID NOs:94-97, 119, 152 and 184-200 show the nucleotide sequences of the MG64 target CRISPR repeat sequences.

[0145] SEQ ID NOs: 237-259 show the nucleotide sequence of MG64 crRNA.

[0146] SEQ ID NOs: 111-114 and 201-204 show the nucleotide sequences of single guide RNAs engineered to function with the MG64 Cas effector.

[0147] SEQ ID NOs: 123, 125, 127, 129, 131, 133 and 153 show the nucleotide sequences of the left-sided transposase recognition sequences associated with the MG64 system.

[0148] SEQ ID NOs: 124, 126, 128, 130, 132, 154 and 155 show the nucleotide sequences of the right transposase recognition sequences associated with the MG64 system.

[0149] SEQ ID NOs: 267-270 show the nucleotide and peptide sequences of the MG64 DNA binding domain CAST fusion proteins.

[0150] MG108

[0151] SEQ ID NOs: 38 and 108 show the full-length peptide sequence of the MG108 Cas effector.

[0152] SEQ ID NOs: 39-40 and 109-110 show peptide sequences of MG108 transposase proteins, which may include a recombinase or transposase complex associated with an MG108 Cas effector. -A, -B, -C, and -Q are added to the ends of the tags to indicate similarity to the TnsA, TnsB, TnsC, and TniQ proteins of the Tn7-like system, respectively.

[0153] SEQ ID NOs:98 and 120 show the nucleotide sequence of the MG108 target CRISPR repeat sequence.

[0154] SEQ ID NOs:260-261 show the nucleotide sequence of MG108 crRNA.

[0155] SEQ ID NOs: 115-116 and 205-206 show the nucleotide sequences of single guide RNAs engineered to function with the MG108 Cas effector.

[0156] SEQ ID NOs: 118, 182-183, and 235-236 show the nucleotide sequences of the MG108 tracrRNA derived from the same locus as the MG108 effector.

[0157] SEQ ID NO: 134 shows the nucleotide sequence of the left-side transposase recognition sequence associated with the MG108 system.

[0158] SEQ ID NO: 135 shows the nucleotide sequence of the right transposase recognition sequence associated with the MG108 system.

[0159] MG110

[0160] SEQ ID NOs: 41-43 and 48-50 show the full length peptide sequences of the MG110 Cas effectors. The addition of -6, -7 and -8 at the ends of the tags indicates similarity to the cas6, cas7 and cas8 proteins of the class I IF system, respectively.

[0161] SEQ ID NOs: 44-47 and 51-54 show peptide sequences of MG110 transposase proteins, which may include a recombinase or transposase complex associated with an MG110 Cas effector. -A, -B, -C and -Q are added to the ends of the tags to indicate similarity to the TnsA, TnsB, TnsC and TniQ proteins of the Tn7-like system, respectively.

[0162] SEQ ID NOs:99-100 show the nucleotide sequences of the MG110 target CRISPR repeat sequences.

[0163] SEQ ID NOs: 121-122 and 207-208 show the nucleotide sequence of MG110 crRNA.

[0164] SEQ ID NOs: 136 and 138 show the nucleotide sequences of the left-side transposase recognition sequences associated with the MG110 system.

[0165] SEQ ID NOs: 137 and 139 show the nucleotide sequences of the right transposase recognition sequences associated with the MG110 system.

[0166] MG161

[0167] SEQ ID NOs: 271-329 show the peptide sequences of the functional domains of MG161.

[0168] MG162

[0169] SEQ ID NOs: 330-340 show the peptide sequences of the functional domains of MG162.

[0170] MG190

[0171] SEQ ID NOs: 341-506 show the peptide sequences of MG190 ribosomal protein.

[0172] Other sequences

[0173] SEQ ID NOs: 140-141 show the peptide sequences of nuclear localization signals.

[0174] SEQ ID NOs: 142-143 show the peptide sequences of the linkers.

[0175] SEQ ID NOs: 144-146 show the peptide sequences of the epitope tags.

[0176] SEQ ID NOs: 264-266 show the peptide sequences of the DNA binding domain. DETAILED DESCRIPTION

[0177] 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.

[0178] 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, 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)).

[0179] 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."

[0180] 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" may mean within one or more than one standard deviation, as practiced in the art. Alternatively, "about" may mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0181] 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, 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 patens, 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).

[0182] 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 derivatives thereof. 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 including 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 marked 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).

[0183] 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 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 defined according to linkage analysis (locus), 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, free polynucleotides including free DNA (cfDNA) and free RNA (cfRNA), nucleic acid probes and primers. The sequence of nucleotides may be interspersed with non-nucleotide components.

[0184] The term "transfection" or "transfected" refers to the introduction of nucleic acid into a cell by a non-viral or viral-based method. 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.

[0185] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues joined 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 both D-amino acids and L-amino acids.

[0186] 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.

[0187] 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 basic elements that promote the transcriptional expression of an operably linked polynucleotide. A eukaryotic basal promoter may contain a TATA box or a CAAT box. In certain embodiments, different promoters guide 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.

[0188] As used herein, the term "expression" refers to the process of transcribing a nucleic acid sequence or polynucleotide from a DNA template (e.g., transcribed 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.

[0189] 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 (e.g., movement or activation) of a first genetic element has some influence on a 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. The influence on the second element can be but need not be the same type as the operation of the first element. For example, if the movement of the first element causes the activation of the second element, the two elements are operably connected. For example, if a regulating element helps to start the transcription of a coding sequence, a regulating element that can include a promoter and / or enhancer sequence is operably connected to the coding region. As long as this functional relationship is maintained, there can be an insertion residue between the regulating element and the coding region.

[0190] 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 a polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. Vectors typically include genetic elements (e.g., regulatory elements) that are operably linked to a gene to promote expression of the gene in a target.

[0191] 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 regulatory elements and one or more genes to which they are operably linked for expression.

[0192] 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.

[0193] 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 non-naturally occurring polynucleotides or polypeptides. Engineered peptides 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 naturally occurring human proteins. For example, the VPR and VP64 domains are synthetic transactivation domains. According to non-limiting examples: nucleic acids can be modified by changing their sequence to a sequence that does not exist in nature; nucleic acids can be modified by connecting them to nucleic acids that are not associated with them in nature, so that the connection product has a function that does not exist in the original nucleic acid; engineered nucleic acids can be synthesized in vitro with sequences that do not exist in nature; proteins can be modified by changing their amino acid sequences to sequences that do not exist in nature; engineered proteins can acquire new functions or properties. An "engineered" system includes at least one engineered component.

[0194] 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 guides 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., from Streptococcus pyogenes (S.pyogenes), Staphylococcus aureus (S.aureus), etc. or SEQ ID NO: *_* tracrRNA). 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 a portion of a repeat sequence in an adjacent CRISPR array.

[0195] As used herein, "guide nucleic acid" or "guide polynucleotide" refers to a nucleic acid that can hybridize with a target nucleic acid and thereby guide 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 inclusive, 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 a sub-segment, which may be referred to as a "protein binding segment" or a "protein binding sequence" or a "Cas protein binding segment."

[0196] 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.

[0197] The terms "sequence identity" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, for example: BLASTP for polypeptide sequences longer than 30 residues using parameters of a wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix with gap penalties set to 11 for presence, 1 for extension, and adjusted using a conditional composition scoring matrix; BLASTP for sequences less than 30 residues using parameters of a wordlength (W) of 2, an expectation (E) of 1000000, and a PAM30 scoring matrix with gap penalties set to 9 for gap opening and 1 for extension (these are the default parameters for BLASTP in the BLAST suite, available at https: / / blast.ncbi.nlm.nih.gov); the Smith-Waterman homology search algorithm using parameters of matches of 2, mismatches of -1, and gaps of -1. CLUSTALW with default searchalgorithm parameters; MUSCLE with default parameters; MAFFT with retree of 2 and maximum iterations of 1000; Novafold with default parameters; HMMER hmmalign with default parameters.

[0198] 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 locating the amino acid residues (e.g., non-conservative residues) that have mutated between species without changing the basic function of the encoded protein. Such conservatively substituted variants may include variants with 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. In some embodiments, such conservatively substituted variants are functional variants. Such functional variants may encompass sequences with substitutions so that the activity of the key active site residues of the endonuclease is not destroyed. In some embodiments, the functional variants of any system in the systems described herein lack substitutions of at least one of the conservative or functional residues shown in Figures 4 or 5. In some embodiments, functional variants of any of the systems described herein lack all substitutions in the conserved or functional residues shown in FIG. 4 and FIG. 5 .

[0199] 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:

[0200] 1) Alanine (A), glycine (G);

[0201] 2) Aspartic acid (D), glutamic acid (E);

[0202] 3) Asparagine (N), glutamine (Q);

[0203] 4) Arginine (R), Lysine (K);

[0204] 5) Isoleucine (I), leucine (L), methionine (M), valine (V);

[0205] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0206] 7) serine (S), threonine (T); and

[0207] 8) Cysteine ​​(C), methionine (M).

[0208] As used herein, the term "RuvC_III domain" refers to the third discontinuous segment of the RuvC nuclease domain (the RuvC nuclease domain comprises 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).

[0209] 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).

[0210] 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.

[0211] As used herein, in the context of nucleic acid modification (e.g., genome 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, for example, excision, insertion, inversion, exchange or translocation of nucleic acid sequences in or between one or more nucleic acid molecules.

[0212] 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 position 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 can be carried out by a cut and paste mechanism or a copy transposition mechanism.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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). Such effector subtypes are typically associated with CAST systems.

[0218] As used herein, the term "IF type" (or Class I, IF type CRISPR) refers to a subtype of a Class I type I CRISPR system. Such systems typically include a multi-component CRISPR effector comprising Cas8, Cas7, and Cas6 proteins. In some cases, such systems are found to be associated with CAST systems. In some cases, the IF type CRISPR system includes crRNA, the crRNA including an 8-nt 5' handle for Cas8 and / or Cas5 binding, a 32-nt spacer for target identification by 7 copies of six Cas, or a 20-nt 3' hairpin for Cas6 binding and pre-crRNA processing. In some cases, the F type system utilizes a 5'-CC PAM on a non-target strand for target binding.

[0219] As used herein, the term "functional domain (FD)" refers to a small protein that can promote protein-DNA interaction. Types of functional domains include, but are not limited to, DNA binding domains ("DBDs") and chromatin regulatory domains ("CMDs"). Non-limiting examples of functional domains include human histone 1 central globular domain (H1 core), high mobility group nucleosome binding domain 1 (HMGN1), chromosome cassette 5 (Cbx5), and Sulfolobus solfataricus sso7d. In some embodiments, the functional domains described herein may be included in a fusion protein or a component thereof having the system described herein. In some embodiments, the fusion protein may exhibit enhanced activity in a cell compared to a non-fusion protein.

[0220] In accordance with IUPAC conventions, the following abbreviations are used in various examples:

[0221] A = Adenine

[0222] C = cytosine

[0223] G = Guanine

[0224] T = thymine

[0225] R = adenine or guanine

[0226] Y = cytosine or thymine

[0227] S = guanine or cytosine

[0228] W = adenine or thymine

[0229] K = guanine or thymine

[0230] M = adenine or cytosine

[0231] B=C, G or T

[0232] D=A, G or T

[0233] H=A、C or T

[0234] V=A、C or G

[0235] Overview

[0236] 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, there are relatively few functionally characterized CRISPR / Cas enzymes 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.

[0237] The CRISPR / Cas system is an RNA-guided nuclease complex that has been described as acting as an adaptive immune system in microorganisms. In its natural environment, the CRISPR / Cas system occurs in a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) operator or locus, which generally includes two parts: (i) an array of short repeat sequences (30-40 bp) separated by equally short spacer sequences, which encode RNA-based targeting elements; and (ii) an ORF encoding a Cas that encodes a nuclease polypeptide guided by the RNA-based targeting elements as well as an auxiliary protein / enzyme. Efficient nuclease targeting of a specific target nucleic acid sequence generally requires both: (i) complementary hybridization between the first 6-8 nucleic acids of the target (target seed) and the crRNA guide; and (ii) the presence of a protospacer adjacent motif (PAM) sequence within a defined vicinity of the target seed (the PAM is typically a sequence that is not commonly represented within the host genome). Depending on the exact function and organization of the system, CRISPR systems are generally divided into 2 categories, 5 types, and 16 subtypes based on shared functional properties and evolutionary similarities (see Figure 1 ).

[0238] Class 1 CRISPR-Cas systems have large multi-subunit effector complexes and include types I, III, and IV.

[0239] Type I CRISPR-Cas systems are considered to have medium complexity in terms of components. In type I CRISPR-Cas systems, the array of RNA targeting elements is transcribed into long precursor crRNA (pre-crRNA), which is processed at the repeat element to release short mature crRNA, when followed by a suitable short consensus sequence called protospacer adjacent motif (PAM), the short mature crRNA guides the nuclease complex to the nucleic acid target. This treatment is carried out by the endoribonuclease subunit (Cas6) of the large endonuclease complex called cascade, and the endonuclease complex also includes the nuclease (Cas3) protein component of the nuclease complex guided by crRNA. Cas I nuclease mainly serves as DNA nuclease.

[0240] 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).

[0241] 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.

[0242] Class 2 CRISPR-Cas systems typically have single-polypeptide multi-domain nuclease effectors and include types II, V, and VI.

[0243] Type II CRISPR-Cas systems are considered to be the simplest in terms of components. In type II CRISPR-Cas systems, the CRISPR array is processed into mature crRNA without the presence of special endonuclease subunits, but requires a small trans-encoded crRNA (tracrRNA), whose region is complementary to the array repeat sequence; tracrRNA interacts with its corresponding effector nuclease (e.g., Cas9) and repeat sequence to form a precursor dsRNA structure, which is cut by endogenous RNase III to generate a mature effector enzyme for both load tracrRNA and crRNA. Cas II nucleases are recorded as DNA nucleases. Type 2 effectors generally show a structure including a RuvC-like endonuclease domain, which adopts an RNase H fold, 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.

[0244] 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 recorded 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.

[0245] Type VI CRIPSR-Cas systems have RNA-guided RNA endonucleases. The single polypeptide effector of type VI systems (e.g., Cas13) includes two HEPN ribonuclease domains, rather than RuvC-like domains. Unlike type II and type V systems, type VI systems do not seem to require tracrRNA to process pre-crRNA into crRNA in some cases. 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 target RNA.

[0246] 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.

[0247] One of the early applications of such a system for in vitro use involved (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 with an approximately 20 nt 5' sequence complementary to the desired target DNA sequence 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 ).

[0248] 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).

[0249] Transposons are mobile elements that can move between positions 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, the most notable example being the Tn7 family.

[0250] 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.

[0251] 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 elements 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.

[0252] Functional domain fusion

[0253] The lack of activity of certain enzyme effectors in human cells may be due to their inability to access the target site, for example, due to compact chromatin structure. Therefore, fusion of functional domains to effectors can improve enzyme activity. For example, fusion of Taq polymerase to sso7d (ds) DNA binding protein improves the processivity of the enzyme, requiring less enzyme and shorter extension time (Wang et al., 2004). Similarly, when fused with various functional domains, CjCas9 has improved cutting and editing efficiency in K562 cells (Ding et al., 2019).

[0254] MG36 System

[0255] In some embodiments, provided herein is a MG36 system for transposing a cargo nucleotide sequence into a target nucleic acid site. Figures 4A-4C. In some embodiments, the system comprises a double-stranded nucleic acid. In some embodiments, the cargo nucleotide sequence is configured to interact with a recombinase complex. In some embodiments, the system comprises a Cas effector complex. In some embodiments, the Cas effector complex comprises 2 types of type II Cas effectors and at least one engineered guide polynucleotide configured to hybridize with a target nucleic acid site. In some embodiments, the 2 types of type II Cas effectors comprise a RuvC domain and a HNH domain. In some embodiments, the system comprises a recombinase or transposase complex, wherein the recombinase or transposase complex is configured to recruit the cargo nucleotide sequence to the target nucleic acid site.

[0256] 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.

[0257] 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.

[0258] In some cases, the engineered guide polynucleotide is configured to bind to the 2 class II type Cas effectors. In some cases, the 2 class II type Cas effectors include a polypeptide 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. In some cases, the 2 class II type Cas effectors include a polypeptide comprising a sequence having at least about 70% identity to SEQ ID NO: 1. In some cases, 2 class II type Cas effectors include polypeptides comprising a sequence having at least about 75% identity to SEQ ID NO: 1. In some cases, 2 class II type Cas effectors include polypeptides comprising a sequence having at least about 80% identity to SEQ ID NO: 1. In some cases, 2 class II type Cas effectors include polypeptides comprising a sequence having at least about 85% identity to SEQ ID NO: 1. In some cases, 2 class II type Cas effectors include polypeptides comprising a sequence having at least about 90% identity to SEQ ID NO: 1. In some cases, 2 class II type Cas effectors include polypeptides comprising a sequence having at least about 91% identity to SEQ ID NO: 1. In some cases, 2 class II type Cas effectors include polypeptides comprising a sequence having at least about 92% identity to SEQ ID NO: 1. In some cases, 2 class II type Cas effectors include polypeptides comprising a sequence having at least about 93% identity to SEQ ID NO: 1. In some cases, 2 class II type Cas effectors include polypeptides comprising a sequence having at least about 94% identity to SEQ ID NO: 1. In some cases, 2 class II type Cas effectors include polypeptides comprising a sequence having at least about 95% identity to SEQ ID NO: 1. In some cases, 2 class II type Cas effectors include polypeptides comprising a sequence having at least about 96% identity to SEQ ID NO: 1. In some cases, 2 class II type Cas effectors include polypeptides comprising a sequence having at least about 97% identity to SEQ ID NO: 1. In some cases, 2 class II type Cas effectors include polypeptides comprising a sequence having at least about 98% identity to SEQ ID NO: 1. In some cases, 2 class II type Cas effectors include polypeptides comprising a sequence having at least about 99% identity to SEQ ID NO: 1.In some cases, the Class 2 type II Cas effector includes a polypeptide comprising a sequence with 100% identity to SEQ ID NO:1.

[0259] In some cases, the recombinase or 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 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: 2-5. In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having at least about 91% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having at least about 92% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having at least about 93% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having at least about 94% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 2-5.In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex includes at least one polypeptide comprising a sequence having 100% identity to any one of SEQ ID NOs: 2-5.

[0260] In some cases, the recombinase or transposase complex comprises 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: 2-5. In some cases, the recombinase or transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 75% identity with any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 80% identity with any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 85% identity with any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 90% identity with any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 91% identity with any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 92% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 93% identity to any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 94% identity to any one of SEQ ID NOs: 2-5.In some cases, the recombinase or transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 95% identity with any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 96% identity with any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 97% identity with any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 98% identity with any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 99% identity with any one of SEQ ID NOs: 2-5. In some cases, the recombinase or transposase complex includes at least a first polypeptide and a second polypeptide, each independently comprising a sequence having 100% identity to any one of SEQ ID NOs: 2-5.

[0261] In some embodiments, the systems disclosed herein include at least one engineered guide polynucleotide (e.g., gRNA).

[0262] In some embodiments, provided herein are engineered guide polynucleotides, such as guide RNA (gRNA).

[0263] In some cases, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides having at least about 70% identity to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides that are at least about 75% identical to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides that are at least about 80% identical to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides that are at least about 85% identical to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides that are at least about 90% identical to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides that are at least about 91% identical to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides that are at least about 92% identical to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides that are at least about 93% identical to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides that are at least about 94% identical to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides that are at least about 95% identical to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides that are at least about 96% identical to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides that are at least about 97% identical to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 consecutive nucleotides that are at least about 98% identical to SEQ ID NO: 11.In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 contiguous nucleotides that is at least about 99% identical to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 contiguous nucleotides that is 100% identical to SEQ ID NO: 11.

[0264] 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.

[0265] 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 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.

[0266] 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.

[0267] 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 replacement 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 a non-connected 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.

[0268] 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, 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 the 2' position, 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).

[0269] 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.

[0270] In some cases, the guide RNA includes a sequence complementary to a eukaryotic, fungal, plant, mammalian or human genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a eukaryotic genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a fungal genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a plant genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a mammalian genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a human genome polynucleotide sequence.

[0271] 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.

[0272] 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 any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence includes a sequence with at least about 80% identity to any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence includes a sequence with at least about 85% identity to any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence includes a sequence with at least about 90% identity to any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence includes a sequence with at least about 91% identity to any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence includes a sequence with at least about 92% identity to any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence includes a sequence with at least about 93% identity to any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence includes a sequence with at least about 94% identity to any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence includes a sequence with at least about 95% identity to any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence includes a sequence with at least about 96% identity to any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence includes a sequence with at least about 97% identity to any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence includes a sequence with at least about 98% identity to any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence includes a sequence with at least about 99% identity to any one of SEQ ID NOs: 17-18. In some cases, the left recombinase sequence includes a sequence with 100% identity to any one of SEQ ID NOs: 17-18.

[0273] 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 NO: 19. In some cases, the right recombinase sequence comprises a sequence having at least about 70% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence comprises a sequence having at least about 75% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence comprises a sequence having at least about 80% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence includes a sequence having at least about 85% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence includes a sequence having at least about 90% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence includes a sequence having at least about 91% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence includes a sequence having at least about 92% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence includes a sequence having at least about 93% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence includes a sequence having at least about 94% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence includes a sequence having at least about 95% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence includes a sequence having at least about 96% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence includes a sequence having at least about 97% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence comprises a sequence having at least about 98% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence comprises a sequence having at least about 99% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence comprises a sequence having 100% identity to SEQ ID NO: 19.

[0274] In some cases, the Class 2 Type II Cas effector and recombinase or 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.

[0275] MG39 System

[0276] In some embodiments, provided herein is the MG39 system for transposing a cargo nucleotide sequence into a target nucleic acid site. Figure 5A-5B , in some embodiments, the system comprises a double-stranded nucleic acid. In some embodiments, the cargo nucleotide sequence is configured to interact with a Tn7-type transposase complex. In some embodiments, the system comprises a Cas effector complex. In some embodiments, the Cas effector complex comprises 2 types of V-type Cas effectors and an engineered guide polynucleotide configured to hybridize with a target nucleotide sequence. In some embodiments, the 2 types of V-type Cas effectors comprise a RuvC domain. In some embodiments, the system comprises a Tn7-type transposase complex configured to bind to a Cas effector complex, wherein the Tn7-type transposase complex comprises a TnsA subunit.

[0277] 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.

[0278] 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.

[0279] In some cases, the engineered guide polynucleotide is configured to bind to a 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: 6. In some cases, the Class 2 type V Cas effector includes a polypeptide comprising a sequence having at least about 70% identity to SEQ ID NO: 6. In some cases, the 2nd class V-type Cas effector includes a polypeptide comprising a sequence having at least about 75% identity to SEQ ID NO:6. In some cases, the 2nd class V-type Cas effector includes a polypeptide comprising a sequence having at least about 80% identity to SEQ ID NO:6. In some cases, the 2nd class V-type Cas effector includes a polypeptide comprising a sequence having at least about 85% identity to SEQ ID NO:6. In some cases, the 2nd class V-type Cas effector includes a polypeptide comprising a sequence having at least about 90% identity to SEQ ID NO:6. In some cases, the 2nd class V-type Cas effector includes a polypeptide comprising a sequence having at least about 91% identity to SEQ ID NO:6. In some cases, the 2nd class V-type Cas effector includes a polypeptide comprising a sequence having at least about 92% identity to SEQ ID NO:6. In some cases, the 2nd class V-type Cas effector includes a polypeptide comprising a sequence having at least about 93% identity to SEQ ID NO:6. In some cases, the 2nd class V-type Cas effector includes a polypeptide comprising a sequence having at least about 94% identity to SEQ ID NO:6. In some cases, the 2nd class V-type Cas effector includes a polypeptide comprising a sequence having at least about 95% identity to SEQ ID NO:6. In some cases, the 2nd class V-type Cas effector includes a polypeptide comprising a sequence having at least about 96% identity to SEQ ID NO:6. In some cases, the 2nd class V-type Cas effector includes a polypeptide comprising a sequence having at least about 97% identity to SEQ ID NO:6. In some cases, the 2nd class V-type Cas effector includes a polypeptide comprising a sequence having at least about 98% identity to SEQ ID NO:6. In some cases, the 2nd class V-type Cas effector includes a polypeptide comprising a sequence having at least about 99% identity to SEQ ID NO:6.In some cases, the Class 2 type V Cas effector includes a polypeptide comprising a sequence with 100% identity to SEQ ID NO:6.

[0280] 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) that includes 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: 8-10. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 91% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 92% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 93% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 94% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 8-10.In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having 100% identity to any one of SEQ ID NOs: 8-10.

[0281] 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: 8-10. 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 70% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 91% identity to any one of SEQ ID NOs: 8-10. 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 92% identity to any one of SEQ ID NOs: 8-10. 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 93% identity to any one of SEQ ID NOs: 8-10. 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 94% identity to any one of SEQ ID NOs: 8-10.In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 8-10. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, each independently comprising a sequence with 100% identity to any one of SEQ ID NOs: 8-10.

[0282] In some cases, the Tn7-type transposase complex includes a TnsA component comprising 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 NO: 7. In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence that is at least about 70% identical to SEQ ID NO: 7. In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence having at least about 75% identity to SEQ ID NO: 7. In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence having at least about 80% identity to SEQ ID NO: 7. In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence having at least about 85% identity to SEQ ID NO: 7. In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence having at least about 90% identity to SEQ ID NO: 7. In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence having at least about 91% identity to SEQ ID NO: 7. In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence having at least about 92% identity to SEQ ID NO: 7. In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence having at least about 93% identity to SEQ ID NO: 7. In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence having at least about 94% identity to SEQ ID NO: 7. In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence having at least about 95% identity to SEQ ID NO: 7. In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence having at least about 96% identity to SEQ ID NO: 7. In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence having at least about 97% identity to SEQ ID NO: 7. In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence having at least about 98% identity to SEQ ID NO: 7. In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence having at least about 99% identity to SEQ ID NO: 7.In some cases, the Tn7-type transposase complex includes a TnsA component comprising a sequence that is 100% identical to SEQ ID NO:7.

[0283] In some cases, the Tn7-type transposase complex includes a TnsB component 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 NO: 8. In some cases, the Tn7-type transposase complex complex includes a TnsB component comprising a sequence at least about 70% identical to SEQ ID NO: 8. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 75% identity to SEQ ID NO: 8. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 80% identity to SEQ ID NO: 8. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 85% identity to SEQ ID NO: 8. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 90% identity to SEQ ID NO: 8. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 91% identity to SEQ ID NO: 8. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 92% identity to SEQ ID NO: 8. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 93% identity to SEQ ID NO: 8. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 94% identity to SEQ ID NO: 8. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 95% identity to SEQ ID NO: 8. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 96% identity to SEQ ID NO: 8. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 97% identity to SEQ ID NO: 8. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 98% identity to SEQ ID NO: 8. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 99% identity to SEQ ID NO: 8.In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence with 100% identity to SEQ ID NO:8.

[0284] In some cases, the Tn7-type transposase complex includes a TnsC component comprising 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 NO: 9. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence that is at least about 70% identical to SEQ ID NO: 9. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 75% identity to SEQ ID NO: 9. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 80% identity to SEQ ID NO: 9. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 85% identity to SEQ ID NO: 9. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 90% identity to SEQ ID NO: 9. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 91% identity to SEQ ID NO: 9. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 92% identity to SEQ ID NO: 9. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 93% identity to SEQ ID NO: 9. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 94% identity to SEQ ID NO: 9. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 95% identity to SEQ ID NO: 9. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 96% identity to SEQ ID NO: 9. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 97% identity to SEQ ID NO: 9. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 98% identity to SEQ ID NO: 9. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 99% identity to SEQ ID NO: 9.In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence with 100% identity to SEQ ID NO:9.

[0285] In some cases, the Tn7-type transposase complex includes a TniQ 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 SEQ ID NO: 10. In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence at least about 70% identical to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence at least about 75% identical to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence that is at least about 80% identical to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence that is at least about 85% identical to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence that is at least about 90% identical to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence that is at least about 91% identical to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence that is at least about 92% identical to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence that is at least about 93% identical to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence that is at least about 94% identical to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence that is at least about 95% identical to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence that is at least about 96% identical to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence that is at least about 97% identical to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence that is at least about 98% identical to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence that is at least about 99% identical to SEQ ID NO: 10.In some cases, the Tn7-type transposase complex includes a TniQ polypeptide comprising a sequence that is 100% identical to SEQ ID NO:10.

[0286] In some embodiments, the systems disclosed herein include at least one engineered guide polynucleotide (e.g., gRNA).

[0287] In some embodiments, provided herein are engineered guide polynucleotides, such as guide RNA (gRNA).

[0288] In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides having at least about 70% identity to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that are at least about 75% identical to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that are at least about 80% identical to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that are at least about 85% identical to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that are at least about 90% identical to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that are at least about 91% identical to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that are at least about 92% identical to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 93% identical to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 94% identical to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 95% identical to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 96% identical to any one of SEQ ID NOs: 13-16.In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 97% identical to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 98% identical to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 99% identical to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is 100% identical to any one of SEQ ID NOs: 13-16.

[0289] 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.

[0290] 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 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.

[0291] 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.

[0292] 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 replacement 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 a non-connected 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.

[0293] 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, 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 the 2' position, 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).

[0294] 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 disaccharide. In some embodiments, the modified sugar includes 2'-O-methoxyethyl. In some embodiments, the guide RNA includes both internucleoside linker modifications and nucleoside modifications.

[0295] In some cases, the guide RNA includes a sequence complementary to a eukaryotic, fungal, plant, mammalian or human genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a eukaryotic genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a fungal genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a plant genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a mammalian genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a human genome polynucleotide sequence.

[0296] 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.

[0297] 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 NO: 20. In some cases, the left recombinase sequence comprises a sequence having at least about 70% identity to SEQ ID NO: 20. In some cases, the left recombinase sequence comprises a sequence having at least about 75% identity to SEQ ID NO: 20. In some cases, the left recombinase sequence comprises a sequence having at least about 80% identity to SEQ ID NO: 20. In some cases, the left recombinase sequence includes a sequence having at least about 85% identity to SEQ ID NO: 20. In some cases, the left recombinase sequence includes a sequence having at least about 90% identity to SEQ ID NO: 20. In some cases, the left recombinase sequence includes a sequence having at least about 91% identity to SEQ ID NO: 20. In some cases, the left recombinase sequence includes a sequence having at least about 92% identity to SEQ ID NO: 20. In some cases, the left recombinase sequence includes a sequence having at least about 93% identity to SEQ ID NO: 20. In some cases, the left recombinase sequence includes a sequence having at least about 94% identity to SEQ ID NO: 20. In some cases, the left recombinase sequence includes a sequence having at least about 95% identity to SEQ ID NO: 20. In some cases, the left recombinase sequence includes a sequence having at least about 96% identity to SEQ ID NO: 20. In some cases, the left recombinase sequence includes a sequence having at least about 97% identity to SEQ ID NO: 20. In some cases, the left recombinase sequence comprises a sequence having at least about 98% identity to SEQ ID NO: 20. In some cases, the left recombinase sequence comprises a sequence having at least about 99% identity to SEQ ID NO: 20. In some cases, the left recombinase sequence comprises a sequence having 100% identity to SEQ ID NO: 20.

[0298] 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 NO: 21. In some cases, the right recombinase sequence comprises a sequence having at least about 70% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence comprises a sequence having at least about 75% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence comprises a sequence having at least about 80% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence includes a sequence having at least about 85% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence includes a sequence having at least about 90% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence includes a sequence having at least about 91% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence includes a sequence having at least about 92% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence includes a sequence having at least about 93% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence includes a sequence having at least about 94% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence includes a sequence having at least about 95% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence includes a sequence having at least about 96% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence includes a sequence having at least about 97% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence comprises a sequence having at least about 98% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence comprises a sequence having at least about 99% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence comprises a sequence having 100% identity to SEQ ID NO: 21.

[0299] 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.

[0300] MG64 System

[0301] In some embodiments, the present invention provides a MG64 system for transposing a cargo nucleotide sequence into a target nucleic acid site. In some embodiments, the system includes 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 includes a Cas effector complex. In some embodiments, the Cas effector complex includes 2 types of V-type Cas effectors and an engineered guide polynucleotide configured to hybridize with a target nucleotide sequence. In some embodiments, the system includes a Tn7-type or Tn5053-type transposase complex configured to bind to a Cas effector complex. In some embodiments, 2 types of V-type Cas effectors include a RuvC domain.

[0302] 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.

[0303] In some cases, the system further includes a target nucleic acid comprising a target nucleic acid site. In some cases, the system further includes 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 the 3' of the target nucleic acid site. In some cases, the PAM sequence is located at the 5' of the target nucleic acid site. In some cases, the PAM sequence includes 5'-nGTn-3' or 5'-nGTt-3'.

[0304] In some cases, the engineered guide polynucleotide is configured to bind to a Class V-type 2 Cas effector. In some cases, the Class V-type 2 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 any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence substantially identical to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147, or a variant thereof. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 91% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 92% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, the Class 2 type V Cas effector includes a polypeptide comprising a sequence at least about 93% identical to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147.In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 94% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, the class 2 type V Cas effector includes a polypeptide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, the class 2 type V Cas effector includes a polypeptide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, the class 2 type V Cas effector includes a polypeptide comprising a sequence having 100% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147.

[0305] 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 polypeptide corresponding to SEQ any of ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150 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. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence at least about 80% identical to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence at least about 85% identical to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence at least about 90% identical to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence at least about 91% identical to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150.In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 92% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 93% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 94% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence at least about 96% identical to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence at least about 97% identical to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence at least about 98% identical to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence at least about 99% identical to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150.In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having 100% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some embodiments, the Tn7-type transposase complex includes TnsB, TnsC, and TniQ.

[0306] 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. 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 70% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. 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 75% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. 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 80% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. 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 85% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, each independently comprising a sequence at least about 90% identical to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150.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 91% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. 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 92% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. 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 93% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. 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 94% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. 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 95% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. 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 96% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, each independently comprising a sequence at least about 97% identical to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150.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 98% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. 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 99% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, each independently comprising a sequence with 100% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150.

[0307] In some cases, the Tn7-type transposase complex includes TnsB, TnsC, and TniQ polypeptides, each of which includes a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150, or variants thereof. In some cases, the Tn7-type transposase complex includes a TnsB polypeptide including a sequence substantially identical to any one of SEQ ID NO: 8 or a variant thereof. In some cases, the Tn7-type transposase complex includes TnsB, TnsC, and TniQ polypeptides, each of which includes a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150, or variants thereof.

[0308] In some cases, the engineered guide polynucleotide comprises 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: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 70% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 75% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 80% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 85% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 90% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 91% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 contiguous nucleotides that are at least about 92% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234.In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 93% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 94% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 95% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 96% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 97% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 98% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 contiguous nucleotides that is at least about 99% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 contiguous nucleotides that is 100% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234.

[0309] In some cases, the engineered guide polynucleotide 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 the non-degenerate nucleotides of any one of SEQ ID NOs: 111-114 and 201-204, or variants thereof. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides substantially identical to the non-degenerate nucleotides of any one of SEQ ID NOs: 111-114 and 201-204, or variants thereof.

[0310] In some cases, the engineered guide polynucleotide comprises 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: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 70% identical to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 75% identical to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 80% identical to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 85% identical to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 90% identical to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 91% identical to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 92% identical to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 93% identical to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 94% identical to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 95% identical to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 96% identical to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 97% identical to any one of SEQ ID NOs: 111-114 and 201-204.In some embodiments, the engineered guide polynucleotide comprises at least about 98% sequence to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises at least about 99% sequence to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises 100% sequence to any one of SEQ ID NOs: 111-114 and 201-204.

[0311] 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.

[0312] 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 replacement 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 a non-connected 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.

[0313] 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, 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 the 2' position, 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).

[0314] 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.

[0315] In some cases, the guide RNA includes a sequence complementary to a eukaryotic, fungal, plant, mammalian or human genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a eukaryotic genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a fungal genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a plant genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a mammalian genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a human genome polynucleotide sequence.

[0316] 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.

[0317] In some cases, the left recombinase sequence comprises 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: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left recombinase sequence comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left recombinase sequence comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left recombinase sequence comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left recombinase sequence comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left recombinase sequence comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left recombinase sequence comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left recombinase sequence comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left recombinase sequence comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left recombinase sequence includes a sequence that is at least about 94% identical to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134.In some cases, the left recombinase sequence comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left recombinase sequence comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left recombinase sequence comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left recombinase sequence comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left recombinase sequence comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left recombinase sequence comprises a sequence having 100% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134.

[0318] 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 any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155. In some cases, the right recombinase sequence comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155. In some cases, the right recombinase sequence comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155. In some cases, the right recombinase sequence comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155. In some cases, the right recombinase sequence comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155. In some cases, the right recombinase sequence comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155. In some cases, the right recombinase sequence comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155. In some cases, the right recombinase sequence comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155. In some cases, the right recombinase sequence comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155. In some cases, the right recombinase sequence comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155. In some cases, the right recombinase sequence includes a sequence that is at least about 95% identical to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155.In some cases, the right recombinase sequence comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155. In some cases, the right recombinase sequence comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155. In some cases, the right recombinase sequence comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155. In some cases, the right recombinase sequence comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155. In some cases, the right recombinase sequence includes a sequence that is 100% identical to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155.

[0319] 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.

[0320] MG108 System

[0321] In some embodiments, provided herein is the MG108 system for transposing a cargo nucleotide sequence into a target nucleic acid site. Figure 8 . 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 transposase complex. In some embodiments, the system comprises a Cas effector complex. In some embodiments, the Cas effector complex comprises 2 types of V-type Cas effectors and an engineered guide polynucleotide configured to hybridize with a target nucleotide sequence. In some embodiments, 2 types of V-type Cas effectors comprise a RuvC domain. In some embodiments, the system comprises a Tn7-type transposase complex configured to bind to a Cas effector complex. In some cases, the Tn7-type transposase complex comprises TnsB and TnsC components but does not include TnsA and / or TniQ components.

[0322] 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.

[0323] 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 3' of the target nucleic acid site.

[0324] In some cases, the engineered guide polynucleotide is configured to bind to a 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: 38 or SEQ ID NO: 108. In some cases, the Class 2 type V Cas effector includes a polypeptide comprising a sequence having at least about 70% identity to SEQ ID NO: 38 or SEQ ID NO: 108. 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: 38 or SEQ ID NO: 108. 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: 38 or SEQ ID NO: 108. 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: 38 or SEQ ID NO: 108. 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 NO: 38 or SEQ ID NO: 108. 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 NO: 38 or SEQ ID NO: 108. 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 NO: 38 or SEQ ID NO: 108. 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 NO: 38 or SEQ ID NO: 108. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 94% identity to SEQ ID NO: 38 or SEQ ID NO: 108. 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: 38 or SEQ ID NO: 108. 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: 38 or SEQ ID NO: 108.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: 38 or SEQ ID NO: 108. 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: 38 or SEQ ID NO: 108. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 99% identity to SEQ ID NO: 38 or SEQ ID NO: 108. In some cases, the class 2 type V Cas effector comprises a polypeptide comprising a sequence having 100% identity to SEQ ID NO: 38 or SEQ ID NO: 108.

[0325] 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) that includes 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: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 91% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 92% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 93% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence at least about 94% identical to any one of SEQ ID NOs: 39-40 and 109-110.In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence that is at least about 99% identical to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence that is 100% identical to any one of SEQ ID NOs: 39-40 and 109-110.

[0326] 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: 39-40 and 109-110. In some cases, 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% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 91% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 92% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 93% identity to any one of SEQ ID NOs: 39-40 and 109-110.In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 94% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, each independently including a sequence having at least about 98% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, each independently including a sequence having at least about 99% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, each independently including a sequence having 100% identity to any one of SEQ ID NOs: 39-40 and 109-110.

[0327] In some cases, the Tn7-type transposase complex includes a TnsB component 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: 40 and 109. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence at least about 70% identical to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 91% identity to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 92% identity to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 93% identity to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 94% identity to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 40 and 109.In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence at least about 97% identical to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence at least about 98% identical to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence at least about 99% identical to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex includes a TnsB component comprising a sequence 100% identical to any one of SEQ ID NOs: 40 and 109.

[0328] In some cases, the Tn7-type transposase complex includes a TnsC component 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: 39 and 110. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence at least about 70% identical to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 91% identity to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 92% identity to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 93% identity to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 94% identity to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 39 and 110.In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence at least about 97% identical to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence at least about 98% identical to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence at least about 99% identical to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex includes a TnsC component comprising a sequence 100% identical to any one of SEQ ID NOs: 39 and 110.

[0329] In some cases, the Tn7-type transposase complex includes TnsB and TnsC components, which respectively include sequences that are 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: 40 and 39 or 109 and 110, or variants thereof. In some cases, the Tn7-type transposase complex includes TnsB and TnsC components, each of which includes a sequence substantially identical to any one of SEQ ID NOs: 40 and 39 or 109 and 110, or a variant thereof.

[0330] In some embodiments, the systems disclosed herein include at least one engineered guide polynucleotide (e.g., gRNA).

[0331] In some embodiments, provided herein are engineered guide polynucleotides, such as guide RNA (gRNA).

[0332] 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: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 70% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 75% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 80% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 85% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 90% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 91% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 92% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 93% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 contiguous nucleotides that are at least about 94% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236.In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 95% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 96% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 97% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 98% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 contiguous nucleotides that is at least about 99% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides that is 100% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236.

[0333] In some cases, the engineered guide polynucleotide 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 the non-degenerate nucleotides of any one of SEQ ID NOs: 115, 116, 205, and 206, or variants thereof. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides substantially identical to the non-degenerate nucleotides of any one of SEQ ID NOs: 115, 116, 205, and 206, or variants thereof.

[0334] In some cases the engineered guide polynucleotide 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 any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 75% identical to any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 80% identical to any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 85% identical to any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 90% identical to any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 91% identical to any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 92% identical to any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 93% identical to any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 94% identical to any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 95% identical to any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 96% identical to any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 97% identical to any one of SEQ ID NOs: 115, 116, 205, and 206.In some embodiments, the engineered guide polynucleotide comprises at least about 98% sequence to any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises at least about 99% sequence to any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that is 100% identical to any one of SEQ ID NOs: 115, 116, 205, and 206.

[0335] 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.

[0336] 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 replacement 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 a non-connected 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.

[0337] 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, 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 the 2' position, 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).

[0338] 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.

[0339] In some cases, the guide RNA includes a sequence complementary to a eukaryotic, fungal, plant, mammalian or human genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a eukaryotic genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a fungal genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a plant genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a mammalian genome polynucleotide sequence. In some cases, the guide RNA includes a sequence complementary to a human genome polynucleotide sequence.

[0340] 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.

[0341] 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 NO: 134. In some cases, the left recombinase sequence comprises a sequence having at least about 70% identity to SEQ ID NO: 134. In some cases, the left recombinase sequence comprises a sequence having at least about 75% identity to SEQ ID NO: 134. In some cases, the left recombinase sequence includes a sequence having at least about 80% identity to SEQ ID NO: 134. In some cases, the left recombinase sequence includes a sequence having at least about 85% identity to SEQ ID NO: 134. In some cases, the left recombinase sequence includes a sequence having at least about 90% identity to SEQ ID NO: 134. In some cases, the left recombinase sequence includes a sequence having at least about 91% identity to SEQ ID NO: 134. In some cases, the left recombinase sequence includes a sequence having at least about 92% identity to SEQ ID NO: 134. In some cases, the left recombinase sequence includes a sequence having at least about 93% identity to SEQ ID NO: 134. In some cases, the left recombinase sequence includes a sequence having at least about 94% identity to SEQ ID NO: 134. In some cases, the left recombinase sequence includes a sequence having at least about 95% identity to SEQ ID NO: 134. In some cases, the left recombinase sequence includes a sequence having at least about 96% identity to SEQ ID NO: 134. In some cases, the left recombinase sequence comprises a sequence having at least about 97% identity to SEQ ID NO: 134. In some cases, the left recombinase sequence comprises a sequence having at least about 98% identity to SEQ ID NO: 134. In some cases, the left recombinase sequence comprises a sequence having at least about 99% identity to SEQ ID NO: 134. In some cases, the left recombinase sequence comprises a sequence having 100% identity to SEQ ID NO: 134.

[0342] 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 NO: 135. In some cases, the right recombinase sequence comprises a sequence having at least about 70% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence comprises a sequence having at least about 75% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence includes a sequence having at least about 80% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence includes a sequence having at least about 85% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence includes a sequence having at least about 90% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence includes a sequence having at least about 91% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence includes a sequence having at least about 92% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence includes a sequence having at least about 93% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence includes a sequence having at least about 94% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence includes a sequence having at least about 95% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence includes a sequence having at least about 96% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence comprises a sequence having at least about 97% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence comprises a sequence having at least about 98% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence comprises a sequence having at least about 99% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence comprises a sequence having 100% identity to SEQ ID NO: 135.

[0343] 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.

[0344] MG110 System

[0345] In some embodiments, the present invention provides an MG110 system for transposing a cargo nucleotide sequence into a target nucleic acid site. In some embodiments, the system includes a double-stranded nucleic acid comprising a cargo nucleotide sequence. In some embodiments, the cargo nucleotide sequence is configured to interact with a Tn7 transposase complex. In some embodiments, the system includes a Cas effector complex. In some embodiments, the Cas effector complex includes a Class I type I Cas effector and an engineered guide polynucleotide configured to hybridize with a target nucleotide sequence. In some embodiments, the system includes a Tn7 transposase complex configured to bind to a Cas effector complex.

[0346] 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.

[0347] 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.

[0348] In some cases, the engineered guide polynucleotide is configured to bind to a Class 1, Type I Cas effector. In some cases, the Class 1, Type I 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 any one of SEQ ID NOs: 41-43 and 48-50. In some cases, the class 1 type I Cas effector includes a polypeptide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, the class 1 type I Cas effector includes a polypeptide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, the class 1 type I Cas effector includes a polypeptide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, the class 1 type I Cas effector includes a polypeptide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, the class 1 type I Cas effector includes a polypeptide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, the class 1 type I Cas effector includes a polypeptide comprising a sequence having at least about 91% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, the class 1 type I Cas effector includes a polypeptide comprising a sequence having at least about 92% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, the class 1 type I Cas effector includes a polypeptide comprising a sequence having at least about 93% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, the class 1 type I Cas effector includes a polypeptide comprising a sequence having at least about 94% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, the class 1 type I Cas effector includes a polypeptide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, the Class 1 type I Cas effector includes a polypeptide comprising a sequence at least about 96% identical to any one of SEQ ID NOs: 41-43 and 48-50.In some cases, the class 1 type I Cas effector comprises a polypeptide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, the class 1 type I Cas effector comprises a polypeptide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, the class 1 type I Cas effector comprises a polypeptide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, the class 1 type I Cas effector comprises a polypeptide comprising a sequence having 100% identity to any one of SEQ ID NOs: 41-43 and 48-50.

[0349] In some cases, the engineered guide polynucleotide is configured to bind to a Class 1, Type I Cas effector. In some cases, the Class 1, Type I Cas effector includes Cas6, Cas7, and Cas8 effectors 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: 41-43 and 48-50. In some cases, the Class 1 Type I Cas effectors include Cas6, Cas7, and Cas8 effectors comprising a sequence substantially identical to any one of SEQ ID NOs: 41-43 and 48-50.

[0350] 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) that includes 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:44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 91% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 92% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 93% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 94% identity to any one of SEQ ID NOs: 44-47 and 51-54.In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least one polypeptide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least one polypeptide including a sequence with 100% identity to any one of SEQ ID NOs: 44-47 and 51-54.

[0351] 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:44-47 and 51-54. In some cases, 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% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, each independently including a sequence having at least about 90% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, each independently including a sequence having at least about 91% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, each independently including a sequence having at least about 92% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex includes at least a first polypeptide and a second polypeptide, each independently including a sequence having at least about 93% identity to any one of SEQ ID NOs: 44-47 and 51-54.In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 94% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex comprises at least a first polypeptide and a second polypeptide, each independently comprising a sequence having 100% identity to any one of SEQ ID NOs: 44-47 and 51-54. In some cases, the Tn7-type transposase complex comprises TnsA, TnsB, TnsC, and TniQ components.

[0352] In some embodiments, the systems disclosed herein include at least one engineered guide polynucleotide (e.g., gRNA).

[0353] In some embodiments, provided herein are engineered guide polynucleotides, such as guide RNA (gRNA).

[0354] In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides having at least about 70% identity to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 75% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 80% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 85% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 90% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 91% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 92% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 93% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 94% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 contiguous nucleotides that are at least about 95% identical to any one of SEQ ID NOs: 121, 122, 207, and 208.In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 96% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 97% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 98% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 consecutive nucleotides that is at least about 99% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 contiguous nucleotides that are 100% identical to any one of SEQ ID NOs: 121, 122, 207, and 208.

[0355] In some cases, the engineered guide polynucleotide 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 any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 75% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 80% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 85% identical to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that is at least about 90% identical to any one o...

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 class II type 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 recombinase or transposase complex configured to bind to the Cas effector complex; c) a double-stranded nucleic acid configured to interact with the recombinase or transposase complex and comprising the cargo nucleotide sequence; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

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

3. The system of claim 1, wherein the Cas effector complex is covalently linked to the recombinase or transposase complex.

4. The system of claim 1, wherein the Cas effector complex is fused to the recombinase or 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 recombinase or 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: 17-18.

7. The system of any one of claim 5, wherein the right recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO:

19.

8. The system of claims 1 to 7, further comprising a PAM sequence that is compatible with the Cas effector complex.

9. The system of claim 8, wherein the PAM sequence is located about 50 to about 70 base pairs from the target nucleic acid site.

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

11. The system of claim 9, wherein the PAM sequence is located 5' to the target nucleic acid site.

12. The system of any one of claims 1 to 11, wherein the Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least 80% identity to SEQ ID NO:

1.

13. The system of any one of claims 1 to 11, wherein the Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least 90% identity to SEQ ID NO:

1.

14. The system of any one of claims 1 to 11, wherein the Class 2 Type II Cas effector comprises a polypeptide comprising the sequence of SEQ ID NO:

1.

15. The system of any one of claims 1 to 14, wherein the recombinase or transposase complex is a Tn7-type transposase complex.

16. The system of any one of claims 1 to 14, wherein the recombinase or transposase complex comprises at least one polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 2-5.

17. The system of any one of claims 1 to 14, wherein the recombinase or transposase complex comprises at least one polypeptide comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 2-5.

18. The system of any one of claims 1 to 17, wherein the recombinase or transposase complex comprises at least one polypeptide comprising the sequence of any one of SEQ ID NOs: 2-5.

19. The system of any one of claims 1 to 18, wherein the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least 80% identity to SEQ ID NO:

12.

20. The system of any one of claims 1 to 18, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to SEQ ID NO:

11.

21. The system of any one of claims 1 to 20, wherein the functional domain is derived from human histone 1 central globular domain, HMGN1, cbx5, or Saccharolobus solfataricus sso7d.

22. The system of any one of claims 1 to 21, wherein the functional domain comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 264-266.

23. The system of any one of claims 1 to 22, wherein the 2 class II type Cas effectors are fused to the functional domain to form a fusion protein.

24. The system of any one of claims 1 to 23, wherein the recombinase or transposase complex comprises a TniQ protein.

25. The system according to claim 24, wherein the TniQ protein is fused to the functional domain to form a fusion protein.

26. The system of claim 25, wherein the TniQ protein comprises a sequence having at least 80% sequence identity to the TniQ domain of SEQ ID NO:

270.

27. The system of any one of claims 1 to 26, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 341-506.

28. The system of any one of claims 1 to 26, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 620, 373, 375, 383, 424, 449, 500, and 506.

29. The system of any one of claims 1 to 28, wherein the 2 class II type Cas effectors and the recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

30. 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 TnsA, TnsB, TnsC and TniQ components; c) a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex and comprising the cargo nucleotide sequence; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

31. The system of claim 30, wherein the Cas effector complex is non-covalently associated with the Tn7-type transposase complex.

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

33. The system of claim 30, wherein the Cas effector complex is fused to the Tn7-type transposase complex.

34. The system of any one of claims 30 to 33, wherein the cargo nucleotide sequence is flanked by a left-side transposase recognition sequence and a right-side transposase recognition sequence recognized by the recombinase or transposase complex.

35. The system of claim 34, wherein the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NO:

20.

36. The system of claim 34, wherein the right recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO:

21.

37. The system of any one of claims 30 to 36, further comprising a PAM sequence that is compatible with the Cas effector complex.

38. The system of claim 37, wherein the PAM sequence is located about 50 to about 70 base pairs from the target nucleic acid site.

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

40. The system of claim 38, wherein the PAM sequence is located 5' to the target nucleic acid site.

41. The system of any one of claims 30 to 40, wherein the Class 2 type V Cas effector is not a Cas12k effector.

42. The system of any one of claims 30 to 41, wherein the TnsA component comprises a polypeptide comprising a sequence having at least 80% identity to SEQ ID NO:

7.

43. The system of any one of claims 30 to 32, wherein the Tn7-type transposase complex comprises at least one polypeptide comprising a sequence at least 80% identical to any one of SEQ ID NOs: 8-10.

44. The system of any one of claims 30 to 43, 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: 13-16.

45. The system of any one of claims 30 to 44, wherein the functional domain is derived from human histone 1 central globular domain, HMGN1, cbx5, or Sulfolobus solfataricus sso7d.

46. ​​The system of any one of claims 30 to 45, wherein the functional domain comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 264-266.

47. The system of any one of claims 30 to 46, wherein the class 2 type V Cas effector is fused to the functional domain to form a fusion protein.

48. The system of claim 47, wherein the fusion protein comprises a sequence at least 80% identical to any one of SEQ ID NOs: 267-268.

49. The system of any one of claims 30 to 48, wherein the Tn7 transposase complex comprises a TniQ protein.

50. The system according to claim 49, wherein the TniQ protein is fused to the functional domain to form a fusion protein.

51. The system of claim 50, wherein the TniQ protein comprises a sequence having at least 80% sequence identity to the TniQ domain of SEQ ID NO:

270.

52. The system of any one of claims 30 to 51, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 341-506.

53. The system of any one of claims 30 to 51, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 620, 373, 375, 383, 424, 449, 500 and 506.

54. The system of any one of claims 30 to 53, wherein the 2 class II type Cas effectors and the recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

55. 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 I IF-type Cas effector, 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 TnsA, TnsB, TnsC and TniQ components; c) a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex and comprising a cargo nucleotide sequence; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

56. The system of claim 55, wherein the Cas effector complex is non-covalently associated with the Tn7-type transposase complex.

57. The system of claim 55, wherein the Cas effector complex is covalently linked to the Tn7-type transposase complex.

58. The system of claim 55, wherein the Cas effector complex is fused to the Tn7-type transposase complex.

59. The system of any one of claims 55 to 58, wherein the cargo nucleotide sequence is flanked by a left-side transposase recognition sequence and a right-side transposase recognition sequence recognized by the recombinase or transposase complex.

60. The system of claim 59, wherein the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 136 and 138.

61. The system of any one of claims 59, wherein the right recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NOs: 137 and 139.

62. The system of claims 55 to 61, further comprising a PAM sequence that is compatible with the Cas effector complex.

63. The system of claim 62, wherein the PAM sequence is located about 50 to about 70 base pairs from the target nucleic acid site.

64. The system of claim 63, wherein the PAM sequence is located 3' to the target nucleic acid site.

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

66. The system of any one of claims 55 to 65, wherein the class I IF-type Cas effector comprises a polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 41-43 and 48-50.

67. The system of any one of claims 55 to 65, wherein the class I IF-type Cas effector comprises a polypeptide comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 41-43 and 48-50.

68. The system of any one of claims 55 to 65, wherein the class I IF-type Cas effector comprises a polypeptide comprising the sequence of any one of SEQ ID NOs: 41-43 and 48-50.

69. The system of any one of claims 55 to 68, wherein the Tn7-type transposase complex comprises at least one polypeptide comprising a sequence at least 80% identical to any one of SEQ ID NOs: 44-47 and 51-54.

70. The system of any one of claims 55 to 68, wherein the Tn7-type transposase complex comprises at least one polypeptide comprising a sequence at least 90% identical to any one of SEQ ID NOs: 44-47 and 51-54.

71. The system of any one of claims 55 to 68, wherein the Tn7-type transposase complex comprises at least one polypeptide comprising the sequence of any one of SEQ ID NOs: 44-47 and 51-54.

72. The system of any one of claims 55 to 71, wherein the functional domain is derived from human histone 1 central globular domain, HMGN1, cbx5, or Sulfolobus solfataricus sso7d.

73. The system of any one of claims 55 to 72, wherein the functional domain comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 264-266.

74. The system of any one of claims 55 to 73, wherein the class I IF-type Cas effector is fused to the functional domain to form a fusion protein.

75. The system of claim 74, wherein the fusion protein comprises a sequence at least 80% identical to any one of SEQ ID NOs: 267-268.

76. The system of any one of claims 55 to 75, wherein the Tn7 transposase complex comprises a TniQ protein.

77. The system of claim 76, wherein the TniQ protein is fused to the functional domain to form a fusion protein.

78. The system of claim 76, wherein the TniQ protein comprises a sequence having at least 80% sequence identity to the TniQ domain of SEQ ID NO:

270.

79. The system of any one of claims 55 to 78, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 341-506.

80. The system of any one of claims 55 to 78, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 620, 373, 375, 383, 424, 449, 500, and 506.

81. The system of any one of claims 55 to 80, wherein the class I IF-type Cas effector and the recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

82. 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) a Class 2 type V Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 90-93, 111-114, 117, 151, 156-181, 201-206, 255, 262, 256, 209, 257, 263, 258, and 210; 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150; 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: 125, 127, 123, 129, 131, 133, 153, and 134; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

83. 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) a class 2 type V Cas effector, the class 2 type V Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 22; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 90, 112, and 202; 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 component comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 23-25; 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, the left recombinase sequence comprising a sequence having at least 80% sequence identity to SEQ ID NO: 125; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 126 and 155; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

84. 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) a class 2 type V Cas effector, the class 2 type V Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 26; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 91, 113, 156, 203, and 209; 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 component comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 27-29; 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, the left recombinase sequence comprising a sequence having at least 80% sequence identity to SEQ ID NO: 127; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NO: 128; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

85. 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) a class 2 type V Cas effector, the class 2 type V Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 60; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 117, 119, 161, and 214; 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 component comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 101-103; 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, the left recombinase sequence comprising a sequence having at least 80% sequence identity to SEQ ID NO: 131; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NO: 132; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

86. 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) a class 2 type V Cas effector, the class 2 type V Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 147; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 151, 152, 181, 234, and 254; 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 component comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 148-150; 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: 153; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NO: 154; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

87. 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) a class 2 type V Cas effector, the class 2 type V Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 34; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 93, 114, 157, 204, and 210; 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 component comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 148-150; 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: 129; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NO: 130; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

88. 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) a class 2 type V Cas effector, the class 2 type V Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 30; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 92, 111, and 201; 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: 31-33; 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: 123; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NO: 124; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

89. 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) a class 2 type V Cas effector, the class 2 type V Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 38; and ii) an engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 98, 115-116, 182, 205-206, and 235; 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: 39 and 40; 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, the left recombinase sequence comprising a sequence having at least 80% sequence identity to SEQ ID NO: 134; ii) the cargo nucleotide sequence; and iii) a right recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NO: 135; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

90. The system of any one of claims 82 to 89, wherein the Class 2 type V Cas effector is a Cas12k effector.

91. The system of any one of claims 82 to 90, further comprising a PAM sequence that is compatible with the Cas effector complex.

92. The system of claim 91, wherein the PAM sequence is located 5' of the target nucleic acid site.

93. The system of any one of claims 91 to 92, wherein the PAM sequence comprises 5'-nGTn-3' or 5'-nGTt-3'.

94. The system of any one of claims 82 to 93, wherein the Cas effector complex further comprises a small prokaryotic ribosomal protein subunit S15.

95. The system of claim 94, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 341-506.

96. The system of claim 94, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 620, 373, 375, 383, 424, 449, 500, and 506.

97. The system of any one of claims 82 to 96, 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.

98. 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 and TnsC components but not TnsA and / or TniQ components; c) a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex and comprising the cargo nucleotide sequence; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

99. The system of claim 98, wherein the Cas effector complex is non-covalently associated with the Tn7-type transposase complex.

100. The system of claim 98, wherein the Cas effector complex is covalently linked to the Tn7-type transposase complex.

101. The system of claim 30, wherein the Cas effector complex is fused to the Tn7-type transposase complex.

102. The system of any one of claims 98 to 101, wherein the cargo nucleotide sequence is flanked by a left transposase recognition sequence and a right transposase recognition sequence recognized by the recombinase or transposase complex.

103. The system of claim 102, wherein the left recombinase sequence comprises a sequence that is at least 80% identical to any one of SEQ ID NO:

134.

104. The system of claim 102, wherein the right recombinase sequence comprises a sequence that is at least 80% identical to SEQ ID NO:

135.

105. The system of any one of claims 98 to 104, further comprising a PAM sequence that is compatible with the Cas effector complex.

106. The system of claim 105, wherein the PAM sequence is located about 50 to about 70 base pairs from the target nucleic acid site.

107. The system of claim 106, wherein the PAM sequence is located 3' to the target nucleic acid site.

108. The system of claim 106, wherein the PAM sequence is located 5' of the target nucleic acid site.

109. The system of any one of claims 98 to 108, wherein the class 2 type V Cas effector is a Cas12k effector.

110. The system of any one of claims 98 to 109, 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: 38 and 108.

111. The system of any one of claims 98 to 109, 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: 38 and 108.

112. The system of any one of claims 98 to 109, wherein the Class 2 type V Cas effector comprises a polypeptide comprising the sequence of any one of SEQ ID NOs: 38 and 108.

113. The system of any one of claims 98 to 112, wherein the TnsB subunit comprises a polypeptide comprising a sequence at least 80% identical to SEQ ID NO:40 or 109.

114. The system of any one of claims 98 to 113, wherein the TnsC subunit comprises a polypeptide comprising a sequence having at least 80% identity to SEQ ID NO:39 or 110.

115. The system of any one of claims 98 to 114, wherein the Tn7-type transposase complex comprises at least one polypeptide comprising a sequence at least 80% identical to any one of SEQ ID NOs: 39-40 and 109-110.

116. The system of any one of claims 98 to 115, wherein the engineered guide polynucleotide comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 115, 116, 205, 206, 261, 235, 260, and 236.

117. The system of any one of claims 98 to 115, 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: 118, 182, 183, 235 and 236.

118. The system of any one of claims 98 to 117, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 341-506.

119. The system of any one of claims 98 to 117, wherein the small prokaryotic ribosomal protein subunit S15 comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 620, 373, 375, 383, 424, 449, 500 and 506.

120. The system of any one of claims 98 to 119, wherein the 2 class II type Cas effectors and the recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

121. A system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a) a Cas effector complex comprising two class II type Cas effectors, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide, wherein the engineered guide polynucleotide is capable of hybridizing to the target nucleic acid; b) a recombinase or transposase complex configured to bind to the Cas effector complex; 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 can be recognized by the recombinase or transposase complex; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

122. 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, the 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 is capable of hybridizing to the target nucleic acid; b) a Tn7-type transposase complex configured to bind to the Cas effector complex and comprising TnsA, TnsB, TnsC and TniQ components; 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 can be recognized by the Tn7-type transposase complex; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

123. 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 I IF type Cas effector, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide, wherein the engineered guide polynucleotide is capable of hybridizing to the target nucleic acid; b) a Tn7-type transposase complex configured to bind to the Cas effector complex and comprising TnsA, TnsB, TnsC and TniQ components; 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 can be recognized by the Tn7-type transposase complex; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

124. 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, the 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 is capable of hybridizing to the target nucleic acid; b) a Tn7-type transposase complex configured to bind to the Cas effector complex and comprising TnsB and TnsC components but not TnsA and / or TniQ components; 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 can be recognized by the Tn7-type transposase complex; and d) Functional domains, including DNA binding domain (DBD) or chromatin regulatory domain (CMD).

125. An engineered nuclease system comprising: an endonuclease comprising a RuvC domain and a HNH domain, wherein the endonuclease is derived from an uncultured microorganism, wherein the endonuclease is a Class 2, Type II endonuclease comprising a sequence having at least 80% identity to SEQ ID NO: 1; as well as An engineered guide polynucleotide, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA includes a spacer sequence configured to hybridize to a target nucleic acid sequence.

126. The engineered nuclease system of claim 125, wherein the engineered guide polynucleotide comprises at least 60-80 contiguous nucleotides having at least 80% identity to SEQ ID NO:

12.

127. The engineered nuclease system of claim 125, wherein the engineered guide polynucleotide comprises a sequence at least 80% identical to SEQ ID NO:

11.

128. 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 V-type endonuclease having at least 80% identity to SEQ ID NO:5; 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.

129. The engineered nuclease system of claim 128, 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: 13-16.

130. 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 endonuclease having at least 80% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147; 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.

131. The engineered nuclease system of claim 130, 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: 90, 91, 92, 93, 117, 151, 156-181, and 209-234.

132. The engineered nuclease system of claim 130 or 131, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 111-114, 201-206, 255, 262, 256, 209, 257, 263, 258, and 210.

133. 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 endonuclease having at least 80% identity to SEQ ID NO: 38 or SEQ ID NO: 108; 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.

134. The engineered nuclease system of claim 133, 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: 118, 182, 183, 235, and 236.

135. The engineered nuclease system of claim 133, wherein the engineered guide polynucleotide comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 111-114 or 201-206, 255, 262, 256, 209, 257, 263, 258, 210, 115, 116, 205, 206, 261, 235, 260, and 236.

136. An engineered nuclease system comprising: A Class I IF-type Cas endonuclease comprising at least one Cas6, Cas7 or Cas8 polypeptide comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 41-43 and 48-50; 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.

137. The engineered nuclease system of claim 136, wherein the engineered guide polynucleotide comprises a sequence at least 80% identical to any one of SEQ ID NOs: 121, 122, 207, and 208.

138. A method for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the method comprising introducing the system according to any one of claims 1 to 137 into a cell.

139. A cell comprising the system of any one of claims 1 to 137.

140. The cell of claim 139, wherein the cell is a eukaryotic cell.

141. The cell of claim 139, wherein the cell is a mammalian cell.

142. The cell of claim 139, wherein the cell is an immortalized cell.

143. The cell of claim 139, wherein the cell is an insect cell.

144. The cell of claim 139, wherein the cell is a yeast cell.

145. The cell of claim 139, wherein the cell is a plant cell.

146. The cell of claim 139, wherein the cell is a fungal cell.

147. The cell of claim 139, wherein the cell is a prokaryotic cell.

148. The cell of claim 139, 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.

149. The cell of claim 139, wherein the cell is an engineered cell.

150. The cell of claim 139, wherein the cell is a stable cell.

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