Fusion proteins

Abstract

The present disclosure provides systems and methods for translocating a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the systems and methods including a double-stranded nucleic acid comprising a cargo nucleotide sequence, the cargo nucleotide sequence 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 a recombinase or transposase complex configured to recruit the cargo nucleotide to the target nucleic acid site.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 313,183, filed February 23, 2022, and U.S. Provisional Patent Application No. 63 / 375,352, filed September 12, 2022, each of which is incorporated in its entirety.

[0002] Sequence Listing 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 their entirety. [Background technology]

[0003] Cas enzymes, along with their associated clustered regularly interspaced short palindromic repeats (CRISPR)-guided ribonucleic acid (RNA), appear to be widespread components of prokaryotic immune systems (approximately 45% of bacteria and 84% of archaea), helping to protect these microorganisms from non-self nucleic acids, such as infectious viruses and plasmids, through CRISPR-RNA-guided nucleic acid cleavage. While deoxyribonucleic acid (DNA) elements encoding CRISPR RNA elements may be relatively conserved in structure and length, their CRISPR-associated (Cas) proteins are highly diverse and contain a wide variety of nucleic acid-interacting domains. While CRISPR DNA elements were observed as early as 1987, the programmable endonuclease cleavage capabilities of CRISPR / Cas complexes have been recognized more recently, leading to the use of recombinant CRISPR / Cas systems in a variety of DNA manipulation and gene editing applications. Summary of the Invention

[0004] In some aspects, the disclosure provides fusion proteins comprising (a) a Class 2 V-type Cas effector and (b) a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD). In some embodiments, the functional domain is derived from the human histone 1 central globular domain, HMGN1, or Saccharolobus solfataricus sso7d. In some embodiments, the Cas effector is derived from the CAST locus. In some embodiments, the Cas effector comprises a sequence having 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% sequence identity to 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 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% sequence identity to any one of SEQ ID NOs: 264-266, or a variant thereof. In some embodiments, the fusion protein comprises a sequence having 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% sequence identity to any one of SEQ ID NOs: 267-268, or a variant thereof.

[0005] 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 modulating domain (CMD). In some embodiments, the TniQ protein is derived from the CAST locus. In some embodiments, the TniQ protein comprises a sequence having 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% sequence identity to the TniQ domain of SEQ ID NO: 270, or a variant thereof. In some embodiments, the functional domain comprises a sequence having 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% sequence identity to any one of SEQ ID NOs: 264-266, or a variant thereof. In some embodiments, the fusion protein comprises a sequence having 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% sequence identity to SEQ ID NO: 270, or a variant thereof.

[0006] In some aspects, the disclosure provides a system for translocating a cargo nucleotide sequence into a target nucleic acid site, the system comprising: a 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 comprising a class 2 type II Cas effector and at least one engineered guide polynucleotide configured to hybridize to the target nucleic acid site; and the recombinase or transposase complex 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 bound 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 Class 2 Type II Cas effector. In some embodiments, the Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having 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 comprising a sequence having at least 80% identity to any one of SEQ ID NOS:2-5, or a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 contiguous 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 having at least 80% identity to SEQ ID NO:11, or a variant thereof. In some embodiments, the left-hand recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOS:17-18, or a variant thereof. In some embodiments, the right-hand recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO:19, or a variant thereof. In some embodiments, the Class 2 Type II Cas effector and the recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

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

[0008] In some aspects, the 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 a class 2 V-type Cas effector and an engineered guide polynucleotide configured to hybridize to the target nucleic acid site; and a Tn7-type transposase complex configured to bind to the Cas effector complex, the Tn7-type transposase complex comprising 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 bound 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 is not a Cas12k effector. 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' to the target nucleic acid site. In some embodiments, the engineered guide polynucleotide is configured to bind to the Class 2 V-type Cas effector. In some embodiments, the TnsA subunit comprises a polypeptide having a sequence at least 80% identical 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 comprising a sequence at least 80% identical to any one of SEQ ID NOs: 8-10, or variants thereof.In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least 80% identity to any one of SEQ ID NOs: 13-16, or a variant thereof. In some embodiments, the left-hand recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 20, or a variant thereof. In some embodiments, the right-hand 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 V-type Cas effector is not a Cas12k effector. In some embodiments, the Class 2 V-type Cas effector and the Tn7-type transposase complex are encoded by polynucleotide sequences comprising less than about 10 kilobases.

[0009] In some aspects, the present disclosure provides a method for translocating a cargo nucleotide sequence into a target nucleic acid site comprising the target nucleotide sequence, comprising expressing in a cell a system of any one of any of the aspects or embodiments described herein or introducing into a cell a system of any one of the aspects or embodiments described herein.

[0010] In some aspects, the disclosure provides methods 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 class 2 type II Cas effector and at least one engineered guide polynucleotide configured to hybridize to 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 is non-covalently bound to the Cas effector complex. In some embodiments, the recombinase or transposase complex is covalently bound 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' 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 Class 2 Type II Cas effector. In some embodiments, the Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having 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 comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 2-5, or variants thereof.In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 contiguous 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 having at least 80% identity to SEQ ID NO: 11, or a variant thereof. In some embodiments, the left-hand recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 17-18, or a variant thereof. In some embodiments, the right-hand recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 19, or a variant thereof. In some embodiments, the class 2 Type II 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 methods 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 class 2 V-type Cas effector and at least one engineered guide polynucleotide configured to hybridize to the target nucleic acid site, a Tn7-type transposase complex configured to bind to the Cas effector complex, the Tn7-type transposase complex comprising a TnsA subunit, and a second double-stranded nucleic acid comprising the target nucleic acid site. In some embodiments, the transposase complex is non-covalently bound to the Cas effector complex. In some embodiments, the transposase complex is covalently bound 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' to the target nucleic acid site. In some embodiments, the engineered guide polynucleotide is configured to bind to the Class 2 V-type Cas effector. In some embodiments, the TnsA subunit comprises a polypeptide having a sequence at least 80% identical 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 comprising a sequence at least 80% identical to any one of SEQ ID NOs: 8-10, or a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least 80% identity to any one of SEQ ID NOs: 13-16, or a variant thereof.In some embodiments, the left-hand recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 20, or a variant thereof. In some embodiments, the right-hand 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 V-type Cas effector is not a Cas12k effector. In some embodiments, the Class 2 V-type Cas effector and the Tn7-type transposase complex are encoded by polynucleotide sequences comprising less than about 10 kilobases.

[0012] In some aspects, the 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 a class I IF-type Cas effector and an engineered guide polynucleotide configured to hybridize to the target nucleic acid site; and a Tn7-type transposase complex configured to bind to the Cas effector complex, the Tn7-type transposase complex comprising 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 bound 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 comprises 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 comprises at least one, at least two, or three polypeptides comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 44-46 or 51-53, or a variant thereof.

[0013] In some aspects, the present disclosure provides a method for translocating a cargo nucleotide sequence into a target nucleic acid site comprising a target nucleotide sequence, comprising expressing in a cell or introducing into a cell a system of any one of the aspects or embodiments described herein.

[0014] In some aspects, the 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 a class 2 V-type Cas effector and an engineered guide polynucleotide configured to hybridize to the target nucleic acid site; and a Tn7-type transposase complex configured to bind to the Cas effector complex, the Tn7-type transposase complex comprising TnsB, TnsC, and TniQ components. and a Cas effector complex, wherein (a) the Class 2 V-type Cas effector comprises a polypeptide having a sequence with 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 with at least 80% sequence identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150, or a variant thereof. In some embodiments, the transposase complex is non-covalently bound to the Cas effector complex. In some embodiments, the transposase complex is covalently bound to the Cas effector complex. In some embodiments, the transposase complex is fused to the Cas effector complex in a single polypeptide. In some embodiments, the Class 2 V-type Cas effector comprises a polypeptide having a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147, or a variant thereof. In some embodiments, the Tn7-type transposase complex comprises a TnsB, TnsC, or TniQ component comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150, or a variant thereof.In some embodiments, the class 2 V-type Cas effector is a Cas12k effector. 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' to 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 V-type Cas effector. In some embodiments, the TnsB, TnsC, and TniQ components each 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 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, or 201-206, 255, 262, 256, 209, 257, 263, 258, 210, or a variant thereof. In some embodiments, the left-hand 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-hand 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 V-type Cas effector and the Tn7-type transposase complex are encoded by polynucleotide sequences 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 to SEQ ID NO: 22, or a variant thereof; (b) the left-hand recombinase sequence comprises a sequence having at least 80% sequence identity to SEQ ID NO: 125, or a variant thereof; (c) the right-hand recombinase sequence comprises a sequence having at least 80% sequence identity to 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 to at least about 46-60 nucleotides of SEQ ID NO: 90, or (ii) comprises a sequence having at least 80% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 94, 112, or 202; or (e) the TnsB, TnsC, and TniQ components comprise a sequence having at least 80% sequence identity to SEQ ID NOs: 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-hand recombinase sequence comprises a sequence having at least 80% sequence identity to SEQ ID NO: 127, or a variant thereof; (c) the right-hand recombinase sequence comprises a sequence having at least 880% sequence identity to SEQ ID NO: 128, or a variant thereof; or (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 NOs: 91, 156, or 209, or (ii) comprises a sequence having at least 80% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 95, 113, or 203, or (e) the TnsB, TnsC, and TniQ components comprise a sequence having at least 80% sequence identity to SEQ ID NOs: 27-29, 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: 60, or a variant thereof; (b) the left-hand recombinase sequence comprises a sequence having at least 80% sequence identity to SEQ ID NO: 131, or a variant thereof; (c) the right-hand recombinase sequence comprises a sequence having at least 80% sequence identity to SEQ ID NO: 132, or a variant thereof; (d) the engineered guide polynucleotide comprises (i) a sequence having at least 80% sequence identity to at least about 46-60 nucleotides of any one of SEQ ID NOs: 117, 161, or 214, or (ii) a sequence having at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 119; or (e) the TnsB, TnsC, and TniQ components comprise a sequence having at least 80% sequence identity to SEQ ID NOs: 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 to SEQ ID NO: 147, or a variant thereof; (b) the left-hand recombinase sequence comprises a sequence having at least 80% sequence identity to SEQ ID NO: 153, or a variant thereof; (c) the right-hand recombinase sequence comprises a sequence having at least 880% sequence identity to SEQ ID NO: 154, 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 NOs: 151, 181, or 234, or (ii) comprises a sequence having at least 80% sequence identity to non-degenerate nucleotides of SEQ ID NO: 152 or 254; or (e) the TnsB, TnsC, and TniQ components comprise a sequence having at least 80% sequence identity to SEQ ID NOs: 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-hand recombinase sequence comprises a sequence having at least 80% sequence identity to SEQ ID NO: 129, or a variant thereof; (c) the right-hand recombinase sequence comprises a sequence having at least 880% sequence identity to SEQ ID NO: 130, or a variant thereof; or (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 NOs: 93, 157, or 210, or (ii) comprises a sequence having at least 80% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 97, 114, or 204, or (e) the TnsB, TnsC, and TniQ components comprise a sequence having at least 80% sequence identity to SEQ ID NOs: 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: 30, or a variant thereof; (b) the left-hand recombinase sequence comprises a sequence having at least 80% sequence identity to SEQ ID NO: 123, or a variant thereof; (c) the right-hand recombinase sequence comprises a sequence having at least 80% sequence identity to SEQ ID NO: 124, or a variant thereof; or (d) the engineered guide polynucleotide comprises (i) a sequence having at least 80% sequence identity to SEQ ID NO: 125, or a variant thereof. (ii) a sequence having at least 80% sequence identity to at least about 46-80 nucleotides of SEQ ID NO: 92, or (iii) a sequence having at least 80% sequence identity to the 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 to SEQ ID NOs: 31, 32, and 33, or variants thereof; or (f) the PAM sequence comprises 5'-nGTn-3' or 5'-nGTt-3'.

[0015] In some aspects, the 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 the cargo nucleotide sequence configured to interact with a Tn7-type transposase complex; a Cas effector complex comprising a class 2 V-type Cas effector and an engineered guide polynucleotide configured to hybridize to the target nucleic acid site; and a Tn7-type transposase complex configured to bind to the Cas effector complex, wherein the Tn7-type transposase complex comprises TnsB and TnsC components but does not comprise 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 bound 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 polypeptide having a sequence 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 having a sequence having at least 80% sequence identity to SEQ ID NO: 40 or 109. In some embodiments, the TnsC component comprises a polypeptide having a sequence having at least 80% sequence identity to SEQ ID NO: 39 or 110. In some embodiments, the Class 2 V-type Cas effector is a Cas12k effector. In some embodiments, the Class 2 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 containing the target nucleic acid site is within a cell.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' to the target nucleic acid site. In some embodiments, the engineered guide polynucleotide is configured to bind to the Class 2 V-type Cas effector. In some embodiments, the TnsB and TnsC components comprise polypeptides having sequences at least 80% identical 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 at least 80% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236, or a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% identity to the non-degenerate nucleotide of any one of SEQ ID NOs: 115, 116, 205, 206, 261, 235, 260, or 236, or a variant thereof. In some embodiments, the left-hand recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 134. In some embodiments, the right-hand recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 135, or a variant thereof. In some embodiments, the class 2 V-type Cas effector and the Tn7-type transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.In some embodiments, (a) the Class 2 V-type Cas effector comprises a sequence having at least 80% sequence identity to SEQ ID NO: 38, or a variant thereof; (b) the left-hand recombinase sequence comprises a sequence having at least 80% sequence identity to SEQ ID NO: 134, or a variant thereof; (c) the right-hand recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO: 135, or a variant thereof; (d) the engineered guide polynucleotide (i) comprises a sequence having at least 80% sequence identity to at least about 46-80 nucleotides of SEQ ID NO: 182 or 235, or (ii) comprises a sequence having at least 80% identity to non-degenerate nucleotides of SEQ ID NOs: 98, 115, 116, 205, and 206; or (e) the TnsB and TnsC components comprise polypeptides having a sequence having at least 80% identity to SEQ ID NOs: 40 and 39, or a variant thereof.

[0016] In some aspects, the disclosure provides an engineered nuclease system comprising: an endonuclease comprising a RuvC domain and an HNH domain, wherein the endonuclease is derived from an uncultured microorganism, and wherein the endonuclease is a Class 2 Type II endonuclease comprising a sequence having at least 80% identity to SEQ ID NO:1, or a variant thereof; and an engineered guide polynucleotide configured to form a complex with the endonuclease, wherein the engineered guide polynucleotide comprises a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered guide polynucleotide comprises at least 60-80 contiguous 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 having at least 80% identity to SEQ ID NO:11, or a variant thereof.

[0017] In some aspects, the disclosure provides an engineered nuclease system comprising an endonuclease comprising a RuvC domain, wherein the endonuclease is derived from an uncultured microorganism, and wherein the endonuclease is a class 2 type V 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 within a target nucleic acid sequence. 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 NOs: 13-16, or a variant thereof.

[0018] In some aspects, the disclosure provides an engineered nuclease system comprising: an endonuclease comprising a RuvC domain, wherein the endonuclease is derived from an uncultured microorganism, and wherein the endonuclease is a Class 2 VK-type endonuclease having at least 80% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147, or a variant thereof; and an engineered guide polynucleotide configured to form a complex with the endonuclease, wherein 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 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, or a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 111-114 or 201-206, 255, 262, 256, 209, 257, 263, 258, 210, or a variant thereof.

[0019] In some aspects, the disclosure provides an engineered nuclease system comprising: an endonuclease comprising a RuvC domain, wherein the endonuclease is derived from an uncultured microorganism, and wherein the endonuclease is a class 2 VK-type endonuclease having at least 80% identity to 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 within a target nucleic acid sequence. 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 a variant thereof. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% identity to the 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 a variant thereof.

[0020] In some aspects, the disclosure provides 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, or a variant thereof; and an engineered guide RNA configured to form a complex with the endonuclease, wherein 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.

[0021] In some aspects, the disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a Cas effector complex comprising a class 2 type II Cas effector, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide configured to hybridize to the target nucleic acid site; a recombinase or transposase complex configured to bind to the Cas effector complex; a double-stranded nucleic acid configured to interact with the recombinase or transposase complex, the double-stranded nucleic acid comprising the cargo nucleotide sequence; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0022] In some embodiments, the Cas effector complex is non-covalently linked 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.

[0023] In some embodiments, the cargo nucleotide sequence is flanked by left and right transposase recognition sequences recognized by a 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.

[0024] In some embodiments, the system further comprises a PAM sequence compatible with a 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.

[0025] In some embodiments, the Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least 80% identity to SEQ ID NO: 1. In some embodiments, the Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, the Class 2 Type II Cas effector comprises a polypeptide comprising the sequence of SEQ ID NO: 1.

[0026] 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 the sequence of any one of SEQ ID NOs: 2-5.

[0027] In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 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.

[0028] In some embodiments, the functional domain is derived from the human histone 1 central globular domain, HMGN1, cbx5, or Saccharolobus 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, a Class 2 Type II Cas effector is fused to the functional domain to form a fusion protein.

[0029] In some embodiments, the recombinase or transposase complex comprises a TniQ protein. In some embodiments, the TniQ protein is fused to a functional domain to form a fusion protein. In some embodiments, the TniQ protein comprises a sequence having at least about 80% sequence identity to the TniQ domain of SEQ ID NO:270.

[0030] 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.

[0031] In some embodiments, the Class 2 Type II Cas effector and recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0032] In some aspects, the disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a Cas effector complex comprising a class 2 V-type Cas effector, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide configured to hybridize to the target nucleic acid site; a Tn7-type transposase complex configured to bind to the Cas effector complex and comprising TnsA, TnsB, TnsC, and TniQ components; a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex and comprising the cargo nucleotide sequence; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0033] 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 bound to the Tn7-type transposase complex. In some embodiments, the Cas effector complex is fused to the Tn7-type transposase complex.

[0034] In some embodiments, the cargo nucleotide sequence is flanked by left and right transposase recognition sequences recognized by a 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.

[0035] In some embodiments, the system further comprises a PAM sequence compatible with a 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.

[0036] In some embodiments, the class 2 V-type Cas effector is not a Cas12k effector.

[0037] In some embodiments, the TnsA component comprises a polypeptide comprising a sequence having at least 80% identity to SEQ ID NO: 7. 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: 8-10.

[0038] 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.

[0039] In some embodiments, the functional domain is derived from human histone 1 central globular domain, HMGN1, cbx5, or Saccharolobus 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, a Class 2 V-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 about 80% identity to any one of SEQ ID NOs: 267-268.

[0040] In some embodiments, the Tn7-type transposase complex comprises a TniQ protein. In some embodiments, the TniQ protein is fused to a functional domain to form a fusion protein. In some embodiments, the TniQ protein comprises a sequence having at least about 80% sequence identity with the TniQ domain of SEQ ID NO: 270.

[0041] 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.

[0042] In some embodiments, the Class 2 Type II Cas effector and recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0043] In some aspects, the disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a Cas effector complex comprising a class 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; a Tn7-type transposase complex configured to bind to the Cas effector complex and comprising TnsA, TnsB, TnsC, and TniQ components; a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex and comprising the cargo nucleotide sequence; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0044] 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 bound to the Tn7-type transposase complex. In some embodiments, the Cas effector complex is fused to the Tn7-type transposase complex.

[0045] In some embodiments, the cargo nucleotide sequence is flanked by left and right transposase recognition sequences recognized by a 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.

[0046] In some embodiments, the system further comprises a PAM sequence compatible with a 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.

[0047] In some embodiments, a 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, a 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, a class I IF-type Cas effector comprises a polypeptide comprising the sequence of any one of SEQ ID NOs: 41-43 and 48-50.

[0048] In some embodiments, a 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, a 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, a Tn7-type transposase complex comprises at least one polypeptide comprising the sequence of any one of SEQ ID NOs: 44-47 and 51-54.

[0049] In some embodiments, the functional domain is derived from human histone 1 central globular domain, HMGN1, cbx5, or Saccharolobus 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, a 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 about 80% identity to any one of SEQ ID NOs: 267-268.

[0050] In some embodiments, the Tn7-type transposase complex comprises a TniQ protein. In some embodiments, the TniQ protein is fused to a functional domain to form a fusion protein.

[0051] In some embodiments, the TniQ protein comprises a sequence having at least about 80% sequence identity to the TniQ domain of SEQ ID NO:270.

[0052] 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.

[0053] In some embodiments, the class I IF-type Cas effector and recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0054] In some aspects, the disclosure provides a system for translocating 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, the Cas effector complex comprising: i) a Class 2 V-type 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) a Class 2 V-type Cas effector comprising a polypeptide having a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 90-93, 11 1-114, 117, 151, 156-181, 201-206, 255, 262, 256, 209, 257, 263, 258, and 210; and a Tn7-type transposase complex configured to bind to the Cas effector complex, the Tn7-type transposase complex comprising TnsB, TnsC, and TniQ components, wherein the TnsB, TnsC, and TniQ components are present. a Tn7-type transposase complex, wherein the TniQ and 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; and a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex, the double-stranded nucleic acid comprising, in 5' to 3' order, i) a sequence selected from the group consisting of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. a double-stranded nucleic acid comprising: ii) a left recombinase sequence comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155; ii) a cargo nucleotide sequence; and iii) a right recombinase sequence comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155; and a functional domain comprising a DNA binding domain (DBD) or a chromatin modulating domain (CMD).

[0055] In some aspects, the disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, comprising: a Cas effector complex configured to hybridize to the target nucleic acid site, the Cas effector complex comprising: i) a 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; and a Tn7-type transposase complex configured to bind to the Cas effector complex, the Tn7-type transposase complex comprising TnsB, TnsC, and TniQ components, wherein the Tn7-type transposase complex binds to the Cas effector complex. The system includes a Tn7-type transposase complex, wherein the sB, TnsC, or TniQ component comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 23-25; a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex, the double-stranded nucleic acid comprising, in 5' to 3' order: i) a left-hand recombinase sequence comprising a sequence having at least 80% sequence identity to SEQ ID NO: 125; ii) a cargo nucleotide sequence; and iii) a right-hand recombinase sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 126 and 155; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0056] In some aspects, the 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, the Cas effector complex comprising: i) a class 2 V-type 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; and a Tn7-type transposer configured to bind to the Cas effector complex, the Tn7-type transposer comprising TnsB, TnsC, and TniQ components. a Tn7-type transposase complex, wherein a TnsB, TnsC, or TniQ component comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 27-29; a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex, the double-stranded nucleic acid comprising, in 5' to 3' order: i) a left-hand recombinase sequence comprising a sequence having at least 80% sequence identity to SEQ ID NO: 127; ii) a cargo nucleotide sequence; and iii) a right-hand recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NO: 128; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0057] In some aspects, the 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, the Cas effector complex comprising: i) a 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; and a Tn7-type transposase complex configured to bind to the Cas effector complex, the Tn7-type transposase complex comprising TnsB, TnsC, and TniQ components. The system includes a Tn7-type transposase complex, wherein the TnsB, TnsC, or TniQ component comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 101-103; a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex, the double-stranded nucleic acid comprising, in 5' to 3' order: i) a left-hand recombinase sequence comprising a sequence having at least 80% sequence identity to SEQ ID NO: 131; ii) a cargo nucleotide sequence; and iii) a right-hand recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NO: 132; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0058] In some aspects, the 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, the Cas effector complex comprising: i) a 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; and a Tn7-type transposer configured to bind to the Cas effector complex, the Tn7-type transposer comprising TnsB, TnsC, and TniQ components. a Tn7-type transposase complex, wherein the TnsB, TnsC, or TniQ component comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 148-150; a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex, the double-stranded nucleic acid comprising, in 5' to 3' order: i) a left-hand recombinase sequence comprising a sequence having at least 80% sequence identity to SEQ ID NO: 153; ii) a cargo nucleotide sequence; and iii) a right-hand recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NO: 154; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0059] In some aspects, the 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, the Cas effector complex comprising: i) a 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; and a Tn7-type transposase configured to bind to the Cas effector complex, the Tn7-type transposase comprising TnsB, TnsC, and TniQ components. The system includes a Tn7-type transposase complex, wherein the TnsB, TnsC, or TniQ component comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 148-150; a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex, the double-stranded nucleic acid comprising, in 5' to 3' order: i) a left-hand recombinase sequence comprising a sequence having at least 80% sequence identity to SEQ ID NO: 129; ii) a cargo nucleotide sequence; and iii) a right-hand recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NO: 130; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0060] In some aspects, the disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, comprising a Cas effector complex configured to hybridize to the target nucleic acid site, the Cas effector complex comprising: i) a 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; and a Tn7-type transposase complex configured to bind to the Cas effector complex, the Tn7-type transposase complex comprising TnsB, TnsC, and TniQ components. the system comprises a Tn7-type transposase complex, wherein the TnsB, TnsC, or TniQ component comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 31-33; a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex, the double-stranded nucleic acid comprising, in 5' to 3' order: i) a left-hand recombinase sequence comprising a sequence having at least 80% sequence identity to SEQ ID NO: 123; ii) a cargo nucleotide sequence; and iii) a right-hand recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NO: 124; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0061] In some aspects, the 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, the Cas effector complex comprising: i) a 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; and a Tn7-type transposon configured to bind to the Cas effector complex, the Tn7-type transposon comprising TnsB, TnsC, and TniQ components. The system includes a Tn7-type transposase complex, wherein the TnsB, TnsC, or TniQ component comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 39 and 40; a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex, the double-stranded nucleic acid comprising, in 5' to 3' order: i) a left-hand recombinase sequence comprising a sequence having at least 80% sequence identity to SEQ ID NO: 134; ii) a cargo nucleotide sequence; and iii) a right-hand recombinase sequence comprising a sequence having at least 80% identity to SEQ ID NO: 135; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0062] In some embodiments, the class 2 V-type Cas effector is a Cas12k effector.

[0063] In some embodiments, the system further comprises a PAM sequence compatible with a Cas effector complex. In some embodiments, the PAM sequence is located 5' to the target nucleic acid site. In some embodiments, the PAM sequence comprises 5'-nGTn-3' or 5'-nGTt-3'.

[0064] 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.

[0065] In some embodiments, the class 2 V-type Cas effector and Tn7-type transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0066] In some aspects, the disclosure provides a system for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, the system comprising: a Cas effector complex comprising a class 2 V-type Cas effector, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide configured to hybridize to the target nucleic acid site; 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; a double-stranded nucleic acid configured to interact with the Tn7-type transposase complex and comprising the cargo nucleotide sequence; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0067] 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 bound to the Tn7-type transposase complex. In some embodiments, the Cas effector complex is fused to the Tn7-type transposase complex.

[0068] In some embodiments, the cargo nucleotide sequence is flanked by left and right transposase recognition sequences recognized by a 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.

[0069] In some embodiments, the right-hand 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 a 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.

[0070] 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 the sequence of any one of SEQ ID NOs: 38 and 108.

[0071] In some embodiments, the TnsB subunit comprises a polypeptide comprising a sequence having at least 80% identity to SEQ ID NO: 40 or 109. In some embodiments, the TnsC subunit comprises a polypeptide comprising a sequence having at least 80% identity to SEQ ID NO: 39 or 110.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] In some embodiments, the Class 2 Type II Cas effector and recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

[0076] In some aspects, the disclosure provides a system for transposing a cargo nucleotide sequence to 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 II Cas effector, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide capable of hybridizing to the target nucleic acid; a recombinase or transposase complex configured to bind to the Cas effector complex; a double-stranded nucleic acid comprising, in 5' to 3' order: i) a left recombinase recognition sequence, ii) a cargo nucleotide sequence, and iii) a right recombinase recognition sequence, wherein the left recombinase recognition sequence and the right recombinase recognition sequence are capable of being recognized by the recombinase or transposase complex; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0077] In some aspects, the disclosure provides a system for transposing a cargo nucleotide sequence to a target nucleic acid site in a target nucleic acid, the system comprising: a Cas effector complex, the Cas effector complex comprising a class 2 V-type Cas effector, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide capable of hybridizing to the target nucleic acid; a Tn7-type transposase complex configured to bind to the Cas effector complex and comprising TnsA, TnsB, TnsC, and TniQ components; a double-stranded nucleic acid comprising, in 5' to 3' order: i) a left-hand recombinase recognition sequence, ii) a cargo nucleotide sequence, and iii) a right-hand recombinase recognition sequence, wherein the left-hand recombinase recognition sequence and the right-hand recombinase recognition sequence are capable of being recognized by the Tn7-type transposase complex; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0078] In some aspects, the disclosure provides a system for transposing a cargo nucleotide sequence to 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 capable of hybridizing to the target nucleic acid; a Tn7-type transposase complex configured to bind to the Cas effector complex and comprising TnsA, TnsB, TnsC, and TniQ components; a double-stranded nucleic acid comprising, in 5' to 3' order: i) a left-hand recombinase recognition sequence, ii) a cargo nucleotide sequence, and iii) a right-hand recombinase recognition sequence, wherein the left-hand recombinase recognition sequence and the right-hand recombinase recognition sequence can be recognized by the Tn7-type transposase complex; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0079] In some aspects, the disclosure provides a system for transposing a cargo nucleotide sequence to a target nucleic acid site in a target nucleic acid, the system comprising: a Cas effector complex, the Cas effector complex comprising a class 2 V-type Cas effector, a small prokaryotic ribosomal protein subunit S15, and an engineered guide polynucleotide capable of hybridizing to the target nucleic acid; a Tn7-type transposase complex configured to bind to the Cas effector complex, the TnsB and TnsC components, but not the TnsA and / or TniQ components; a double-stranded nucleic acid comprising, in 5' to 3' order: i) a left-hand recombinase recognition sequence, ii) a cargo nucleotide sequence, and iii) a right-hand recombinase recognition sequence, wherein the left-hand recombinase recognition sequence and the right-hand recombinase recognition sequence can be recognized by the Tn7-type transposase complex; and a functional domain comprising a DNA-binding domain (DBD) or a chromatin modulating domain (CMD).

[0080] In some aspects, the disclosure provides an engineered nuclease system comprising: an endonuclease comprising a RuvC domain and an HNH domain, wherein the endonuclease is derived from an uncultured microorganism, and wherein the endonuclease is a Class 2 Type II endonuclease comprising a sequence having at least 80% identity to SEQ ID NO:1; and an engineered guide polynucleotide, wherein an 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.

[0081] In some embodiments, the engineered guide polynucleotide comprises at least 60-80 contiguous nucleotides having at least 80% identity to SEQ ID NO:12.

[0082] In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% identity to SEQ ID NO:11.

[0083] Provided is 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 type V 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 wherein the engineered guide RNA comprises a spacer sequence configured to hybridize to a target nucleic acid sequence.

[0084] 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.

[0085] 1. 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 wherein 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: 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.

[0087] In some aspects, the disclosure provides an engineered nuclease system comprising: an endonuclease comprising a RuvC domain, wherein the endonuclease is derived from an uncultured microorganism, and wherein the endonuclease is a class 2 VK-type 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 wherein 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: 118, 182, 183, 235, and 236. In some embodiments, 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.

[0089] In some aspects, the disclosure provides 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. 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.

[0090] In some aspects, the present disclosure provides methods for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, comprising introducing a system of the present disclosure into a cell. In some aspects, the disclosure provides a cell comprising the 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 cells are A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof. In some embodiments, the cells are engineered cells. In some embodiments, the cells are stable cells.

[0091] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. [Brief explanation of the drawings]

[0092] The novel features of the present disclosure are set forth with particularity 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 that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also referred to herein as "Figure" and "FIG.").

[0093] [Figure 1] Illustrates exemplary organizations of different classes and types of CRISPR / Cas loci. [Figure 2] Illustrates the structure of the natural class 2 type II crRNA / tracrRNA pair compared to a hybrid sgRNA in which the crRNA and tracrRNA are joined. [Figure 3] The two pathways found in Tn7 and Tn7-like elements are illustrated. [Figure 4A] The genomic context of the type II Tn7-reduced CAST of the MG36 family is illustrated in Figure 4A. The MG36-5 CAST system contains 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] Figure 4B illustrates the genomic context of type II Tn7-reduced CAST in the MG36 family. Figure 4B shows the two predicted transposon ends for the MG36-1 CAST line (TIR-1 and TIR-2). [Figure 4C] The genomic context of type II Tn7-reduced CAST in the MG36 family is illustrated in Figure 4C. Figure 4C shows an alignment of the predicted type II Tn7-reduced CAST transposon left end (LE) and right end (RE) sequences, with annotated repeats indicated as arrows. The left and right ends are labeled according to their orientation. [Figure 5A] The genomic context of type V Tn7 CAST in the MG39 family is illustrated. Figure 5A shows that the MG39-1 CAST system contains a type V nuclease, four predicted transposon proteins (TnsABC and TniQ), and a CRISPR array. Transposon ends were predicted for the MG39-1 CAST system (TIR-1). [Figure 5B]Figure 5B illustrates the genomic context of type V Tn7 CAST in the MG39 family. Figure 5B shows an alignment of the predicted type V Tn7 CAST transposon left end (LE) and right end (RE) sequences, with annotated inverted repeats represented as arrows. [Figure 6] Illustrated is the predicted structure of the corresponding sgRNA of the CAST system described herein (e.g., as predicted in Example 3). [Figure 7] Illustrated is the predicted structure of the corresponding sgRNA of the CAST system described herein (e.g., as predicted in Example 3). [Figure 8] Illustrates the genomic context of MG108-1, a system described herein. This candidate is a Cas12K CAST that naturally lacks TniQ. Genes in the genomic fragment are represented by arrows. [Figure 9] The phylogenetic gene lineage of Cas12k effector sequences is illustrated. The lineage was inferred from a multiple sequence alignment of 64 Cas12k sequences recovered here (orange and black branches) and 229 reference Cas12k sequences from public databases (gray branch). The orange branch indicates Cas12k effectors confirmed to be associated with CAST transposon components. [Figure 10A] MG110 Cascade CAST is illustrated. Figure 10A shows the genomic context of MG110-1 Cascade CAST. The complete Tn7 suite (TnsA, TnsB, TnsC / TniB, TniQ) and defective Cascade suites (Cas6, Cas7, and fused Cas5-Cas8) are represented by orange arrows. The TIRs flanking the CAST transposon are represented by connected arrows. [Figure 10B] Figure 10B illustrates the MG110 cascade CAST. Figure 10B illustrates the repeating secondary structure showing the stem-loop structure of the crRNA. [Figure 10C]10C illustrates the MG110 cascade CAST. As shown in Figure 10C, a sequence alignment of CRISPR repeats from A. wodanis and V. cholerae is shown, and the MG110 family CAST shows a conserved motif indicative of the crRNA stem-loop secondary structure. [Figure 11A] The MG64-3 CRISPR locus is illustrated. The tracrRNA is encoded upstream from the CRISPR array, while the transposon ends are encoded downstream (inner black box). Sequences corresponding to partial 3' CRISPR repeats and a partial spacer are encoded within the transposon (outer box). A self-matching spacer is encoded outside the transposon ends. [Figure 11B-1] Figure 1 illustrates tracrRNA sequence alignments for various CASTs provided herein. The tracrRNA sequence alignment shows conserved regions. In particular, the sequence "TGCTTTC" (upper box) at sequence positions 92-98 is suggested to be important for sgRNA tertiary structure and for non-contiguous repeat-repeat suppressor pairing with the crRNA. The hairpin "CYCC(n6)GGRG" (lower box) at positions 265-278 may be important for its potential function of positioning downstream sequences for crRNA pairing. [Figure 11B-2] Figure 1 illustrates tracrRNA sequence alignments for various CASTs provided herein. The tracrRNA sequence alignment shows conserved regions. In particular, the sequence "TGCTTTC" (upper box) at sequence positions 92-98 is suggested to be important for sgRNA tertiary structure and for non-contiguous repeat-repeat suppressor pairing with the crRNA. The hairpin "CYCC(n6)GGRG" (lower box) at positions 265-278 may be important for its potential function of positioning downstream sequences for crRNA pairing. [Figure 11C] For example, we show the presence of the repeat-repeat suppression (RAR) motif in the MG64-2, MG64-4, MG64-5, MG64-6, MG64-7, and MG108-1 families. [Figure 12A]The predicted structure of the MG64-2 sgRNA is illustrated. [Figure 12B] The predicted structure of the MG64-4 sgRNA is illustrated. [Figure 12C] The predicted structure of the MG64-6 sgRNA is illustrated. [Figure 12D] The predicted structure of the MG64-7 sgRNA is illustrated. [Figure 12E] The predicted structure of the MG108-1 sgRNA is illustrated. [Figure 13A] Figure 13A illustrates PCR, PAM, and Sanger sequencing data demonstrating that MG64-6 is active in vitro. Using the protocol described for in vitro target integrase activity, the effector protein and its TnsB, TnsC, and TniQ proteins were expressed in an in vitro transcription / translation system. After translation, target DNA, cargo DNA, and sgRNA were added to the reaction buffer. Integration was assayed by PCR across the target / donor junction. Figure 13A illustrates a gel image of transposition PCR showing 64-6 apo (no sgRNA) and 64-6 sgRNA with sgRNA. PCR 3 detects the RE junction, PAM distal. PCR 4 is the LE junction, PAM distal. PCR 5 is the RE junction, PAM proximal. PCR 6 is the LE junction, PAM proximal. PCRs are paired across different possible orientations (PCRs 3 and 6 vs. PCRs 4 and 5). The LE-PAM proximal and RE-PAM distal orientation is preferred. [Figure 13B] Figure 13B illustrates PCR, PAM, and Sanger sequencing data demonstrating that MG64-6 is active in vitro. Using the protocol described for in vitro target integrase activity, the effector protein and its TnsB, TnsC, and TniQ proteins were expressed in an in vitro transcription / translation system. After translation, target DNA, cargo DNA, and sgRNA were added to the reaction buffer. Integration was assayed by PCR across the target / donor junction. Figure 13B illustrates the PAM from the in vitro transposition assay, sequencing PCRs 5 and 6. [Figure 13C-1]Figure 13C-1 illustrates PCR, PAM, and Sanger sequencing data demonstrating that MG64-6 is active in vitro. Using the protocol described for in vitro target integrase activity, the effector protein and its TnsB, TnsC, and TniQ proteins were expressed in an in vitro transcription / translation system. After translation, target DNA, cargo DNA, and sgRNA were added to the reaction buffer. Integration was assayed by PCR across the target / donor junction. Figure 13C-1 illustrates Sanger data showing the translocation junction where excision occurs in the donor DNA. The first panel shows PCRs 3 and 5 (RE). The second panel shows PCRs 4 and 6 (LE). The Sanger sequencing reaction is of the donor target product; therefore, the point at which sequencing stops matching the donor DNA is when the junction occurs (dark bar below the sequencing peak). [Figure 13C-2] Figure 13C-2 illustrates PCR, PAM, and Sanger sequencing data demonstrating that MG64-6 is active in vitro. Using the protocol described for in vitro target integrase activity, the effector protein and its TnsB, TnsC, and TniQ proteins were expressed in an in vitro transcription / translation system. After translation, target DNA, cargo DNA, and sgRNA were added to the reaction buffer. Integration was assayed by PCR across the target / donor junction. Figure 13C-2 illustrates Sanger data showing the translocation junction where excision occurs in the donor DNA. The first panel shows PCRs 3 and 5 (RE). The second panel shows PCRs 4 and 6 (LE). The Sanger sequencing reaction is of the donor target product; therefore, the point at which sequencing stops matching the donor DNA is when the junction occurs (dark bar below the sequencing peak). [Figure 14] Figure 1 illustrates next-generation sequencing (NGS) results of in vitro transposition products revealing insertion site preference. NGS reads were compared to a reference sequence with a transposition at position 60 and processed with CRISPResso2. Indels from this correspond to transpositions earlier or later than this arbitrary reference sequence. [Figure 15]Figure 1 illustrates the results of an electrophoretic mobility shift assay (EMSA) of 64-2 TnsB and its RE DNA sequence. EMSA results confirm 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 is 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 + in vitro transcription / translation system (no TnsB protein). Lane 3: FAM DNA + TnsB. The upshift of the labeled band in lane 3 indicates binding of the RE sequence by TnsB, indicating that it contains an active RE translocation sequence. [Figure 16] The activity of Cas12k and TniQ fusions tested in vitro is depicted in Figure 16. Panel A of Figure 16 depicts a gel image showing the transposition activity of the left end to the donor. Lane 1 = apo (no sgRNA), lane 2 = holo (with sgRNA), lane 3 = MG64-6-Ca12k-sso7d-NLS, lane 4 = NLS-sso7d-MG64-6-Ca12k, lane 5 = MG64-6-Q-H1core-NLS, lane 6 = MG64-6-Q-HMGN1-NLS. Panel B of Figure 16 depicts a gel image showing the 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-H1core-NLS, lane 5 = skip, lane 6 = skip, lane 7 = nuclear MG64-6-Cas12k-sso7d-NLS, lane 8 = nuclear MG64-6-Q-H1core-NLS. [Figure 17] Figure 1 illustrates a phylogeny demonstrating that MG161 family members are distant homologs of sso7d. The phylogeny was inferred from multiple sequence alignments of full-length protein sequences containing the PFam PF02294 domain hits. The reference sso7d sequence is highlighted with a triangle. The tip-to-tip distance is estimated at 0.5 substitutions per site (horizontal bar). [Figure 18A]The genomic context of a protein encoding multiple functional domains (FDs) is illustrated. The FDs correspond to tandem imperfect repeats (arrows labeled 161-12 to 161-18). [Figure 18B] Figure 1 illustrates a multiple sequence alignment of tandem repeat FDs to the reference sso7d sequence from S. solfataricus. MG161-13 has 20% amino acid identity (AAI) to the reference sequence, while the other FDs have lower sequence identity. [Figure 19] Figure 1 illustrates a phylogeny demonstrating that MG162 family members are distant homologs of HMGN1. The phylogeny was inferred from multiple sequence alignments of full-length protein sequences containing the PFam PF01101 domain hits. The reference HMGN1 sequence is highlighted with a triangle. The distance between the extremities is estimated at 0.3 substitutions per site (horizontal bar). [Figure 20] Figure 1 illustrates a multiple sequence alignment of the MG162 functional domain protein versus the reference human and mouse HMGN1 sequences. The average pairwise percent identity of the alignment is 40.4%. The conserved RXSXRLS motif is highlighted in a black box. [Figure 21] Figure 1 illustrates a schematic of the identification of ribosomal protein S15 homologs in cyanobacterial genome fragments. Candidate sequences from the same sample in which the Cas12k effector was recovered are highlighted with dark dots. The reference S15 from E. coli is indicated by an arrow.

[0094] Brief description of the sequence listing 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 are exemplary descriptions of the sequences therein.

[0095] MG36 SEQ ID NO: 1 shows the full-length peptide sequence of the MG36 Cas effector.

[0096] SEQ ID NOS:2-5 show peptide sequences of MG36 transposition proteins, which may comprise a recombinase or transposase complex associated with the MG36 Cas effector. The addition of -B1, -B2, -T1, and -C to the ends of the tags indicates similarity to the TnsB1, TnsB2, TnsT1, and TniC proteins of Tn7-like systems, respectively.

[0097] SEQ ID NO: 11 shows the nucleotide sequence of an sgRNA engineered to function with the MG36 Cas effector.

[0098] SEQ ID NO: 12 shows the nucleotide sequence of the MG36 tracrRNA, which is derived from the same locus as the MG36 Cas effector.

[0099] SEQ ID NOs: 17-18 show the nucleotide sequences of the left transposase recognition sequences associated with the MG36 system.

[0100] SEQ ID NO: 19 shows the nucleotide sequence of the right transposase recognition sequence associated with the MG36 system.

[0101] MG39 SEQ ID NO: 6 shows the full-length peptide sequence of the MG39-1 Cas effector.

[0102] SEQ ID NOs: 7-10 show the peptide sequences of MG39-1 transposition proteins, which may comprise a recombinase or transposase complex associated with the MG39-1 Cas effector.

[0103] SEQ ID NOs: 13-16 show the nucleotide sequences of the MG39 tracrRNA, which is derived from the same locus as the MG39 Cas effector.

[0104] SEQ ID NO: 20 shows the nucleotide sequence of the left-hand transposase recognition sequence associated with the MG39 system.

[0105] SEQ ID NO: 21 shows the nucleotide sequence of the right transposase recognition sequence associated with the MG39 system.

[0106] MG64 SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147 show the full-length peptide sequences of the MG64 Cas effectors.

[0107] SEQ ID NOS: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150 set forth peptide sequences of MG64 transposition proteins, which may comprise a recombinase or transposase complex associated with an MG64 Cas effector. The addition of -A, -B, -C, and -Q to the ends of the tags indicates similarity to the TnsA, TnsB, TnsC, and TniQ proteins of Tn7-like systems, respectively.

[0108] 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.

[0109] SEQ ID NOs: 94-97, 119, 152, and 184-200 show the nucleotide sequences of MG64-targeted CRISPR repeats.

[0110] SEQ ID NOs: 237-259 show the nucleotide sequences of MG64 crRNA.

[0111] SEQ ID NOs: 111-114 and 201-204 show the nucleotide sequences of single guide RNAs engineered to function with the MG64 Cas effector.

[0112] SEQ ID NOs: 123, 125, 127, 129, 131, 133, and 153 show the nucleotide sequences of the left-hand transposase recognition sequences associated with the MG64 system.

[0113] 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.

[0114] SEQ ID NOs: 267 to 270 show the nucleotide and peptide sequences of the MG64 DNA-binding domain CAST fusion protein.

[0115] MG108 SEQ ID NOs: 38 and 108 show the full-length peptide sequence of the MG108 Cas effector.

[0116] SEQ ID NOS:39-40 and 109-110 show peptide sequences of MG108 transposition proteins, which may comprise a recombinase or transposase complex associated with the MG108 Cas effector. The addition of -A, -B, -C, and -Q to the ends of the tags indicates similarity to the TnsA, TnsB, TnsC, and TniQ proteins of Tn7-like systems, respectively.

[0117] SEQ ID NOs: 98 and 120 show the nucleotide sequences of the MG108-targeted CRISPR repeats.

[0118] SEQ ID NOs: 260-261 show the nucleotide sequence of MG108 crRNA.

[0119] SEQ ID NOs: 115-116 and 205-206 show the nucleotide sequences of single guide RNAs engineered to function with the MG108 Cas effector.

[0120] SEQ ID NOs: 118, 182-183, and 235-236 show the nucleotide sequences of MG108 tracrRNAs derived from the same locus as the MG108 effector.

[0121] SEQ ID NO: 134 shows the nucleotide sequence of the left-hand transposase recognition sequence associated with the MG108 lineage.

[0122] SEQ ID NO: 135 shows the nucleotide sequence of the right transposase recognition sequence associated with the MG108 lineage.

[0123] MG110 SEQ ID NOS: 41-43 and 48-50 show the full-length peptide sequences of the MG110 Cas effector. The addition of -6, -7, and -8 to the termini of the tags indicates similarity to the cas6, cas7, and cas8 proteins of class I and IF systems, respectively.

[0124] SEQ ID NOS:44-47 and 51-54 show peptide sequences of MG110 transposition proteins, which may comprise a recombinase or transposase complex associated with an MG110 Cas effector. The addition of -A, -B, -C, and -Q to the ends of the tags indicates similarity to the TnsA, TnsB, TnsC, and TniQ proteins of Tn7-like systems, respectively.

[0125] SEQ ID NOs: 99-100 show the nucleotide sequences of the MG110-targeted CRISPR repeats.

[0126] SEQ ID NOs: 121-122 and 207-208 show the nucleotide sequences of MG110 crRNA.

[0127] SEQ ID NOs: 136 and 138 show the nucleotide sequences of the left-hand transposase recognition sequences associated with the MG110 system.

[0128] SEQ ID NOs: 137 and 139 show the nucleotide sequences of the right transposase recognition sequences associated with the MG110 system.

[0129] MG161 SEQ ID NOs: 271 to 329 show peptide sequences of MG161 functional domains.

[0130] MG162 SEQ ID NOs: 330 to 340 show the peptide sequences of the MG162 functional domains.

[0131] MG190 SEQ ID NOs: 341 to 506 show the peptide sequences of MG190 ribosomal protein.

[0132] Other arrays SEQ ID NOs: 140 to 141 show the peptide sequences of nuclear localization signals.

[0133] SEQ ID NOs: 142 to 143 show the peptide sequences of the linkers.

[0134] SEQ ID NOs: 144 to 146 show the peptide sequences of epitope tags.

[0135] SEQ ID NOs: 264 to 266 show the peptide sequences of the DNA binding domain. DETAILED DESCRIPTION OF THE INVENTION

[0136] While various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0137] The practice of some methods disclosed herein employs, unless otherwise indicated, techniques in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012), the series Current Protocols in Molecular Biology (F.M.A.usubel, et al. eds.), the series Methods in Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J.MacPherson, B.D.Hames and G.R.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 (R.I. Freshney, ed. (2010)).

[0138] 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. Furthermore, to the extent that the terms "comprise," "include," "have," "have," "having," or variants thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0139] 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 depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0140] 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 originate from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, single-celled eukaryotic cells, protozoan cells, cells from plants (e.g., plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, hornworts, bryophytes, mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, etc.), and the like. Examples of cells include cells from various organisms, such as porcine algae (e.g., C. Agardh), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from vertebrates (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. In some cases, the cells are not derived from a naturally occurring organism (e.g., the cells may be synthetically produced and sometimes referred to as artificial cells).

[0141] As used herein, the term "nucleotide" refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may 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 derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide may refer to dideoxyribonucleoside triphosphates (ddNTPs) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled, such as by using a moiety containing an optically detectable moiety (e.g., a fluorophore). Labeling may also be performed using quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, all available from Perkin Elmer (Foster City, Calif.); fluoro-conjugated deoxynucleotides, fluoro-conjugated Cy3-dCTP, fluoro-conjugated Cy5-dCTP, fluoro-conjugated fluoroX-dCTP, fluoro-conjugated Cy3-dUTP, and fluoro-conjugated Cy5-dUTP, all available from Amersham (Arlington Heights, Ill.); and Boehringer 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 Mannheim (Indianapolis, Ind.); and Molecular Examples of chromosomal labeled nucleotides available from Probes (Eugene, Oreg.) include 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).

[0142] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, in single-, double-, or multiple-stranded form. A polynucleotide may be exogenous or endogenous to a cell. A polynucleotide may be present in a cell-free environment. A polynucleotide may be a gene or a fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may have any three-dimensional structure and may perform any function. When referring to a polynucleotide, T means U (uracil) in RNA and T (thymine) in DNA. A polynucleotide may contain one or more analogs (e.g., modified backbones, sugars, or nucleobases). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, heterologous nucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein attached to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosine, and wyosine.Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci (gene loci) defined by binding analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides, including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.

[0143] The term "transfection" or "transfected" refers to the introduction of nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecule can be a gene sequence encoding an entire protein or a functional portion thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0144] 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. The term does not denote a specific length of the polymer, and is not intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or naturally occurring. The term applies to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer is interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins and proteins with or without secondary and / or tertiary structure (e.g., domains). The term also encompasses amino acid polymers modified by any other manipulation, such as disulfide bond formation, glycosylation, lipid formation, acetylation, phosphorylation, oxidation, and conjugation with a labeling component. As used herein, the terms "amino acid" and "amino acids" refer to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids may include natural amino acids and unnatural amino acids, which are chemically modified to include non-naturally occurring groups or chemical moieties on the amino acid. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes both D- and L-amino acids.

[0145] As used herein, "non-naturally occurring" may refer to a nucleic acid or polypeptide sequence that is not found in a naturally occurring nucleic acid or protein. Non-naturally occurring may refer to an affinity tag. Non-naturally occurring may refer to a fusion. Non-naturally occurring may refer to a naturally occurring nucleic acid or polypeptide sequence that includes mutations, insertions, and / or deletions. A non-naturally occurring sequence may exhibit and / or encode an activity (e.g., an enzymatic activity, a methyltransferase activity, an acetyltransferase activity, a kinase activity, a ubiquitination activity, etc.) that may also be exhibited by the nucleic acid and / or polypeptide sequence to which the non-naturally occurring sequence is fused. A non-naturally occurring nucleic acid or polypeptide sequence may be linked to a naturally occurring nucleic acid and / or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid and / or polypeptide sequence that encodes a chimeric nucleic acid or polypeptide.

[0146] As used herein, the term "promoter" refers to a regulatory DNA region that controls the transcription or expression of a polynucleotide (e.g., a gene) and may be located adjacent to or overlapping the nucleotide or region of nucleotide at which RNA transcription is initiated. A promoter may contain specific DNA sequences that bind protein factors, often called transcription factors, which promote the binding of RNA polymerase to DNA, thereby resulting in gene transcription. A "basal promoter," also called a "core promoter," may refer to a promoter that contains all the basic elements required to promote the transcriptional expression of an operably linked polynucleotide. A basal promoter in eukaryotes may contain a TATA box or CAAT box. In some embodiments, different promoters induce the expression of a gene in different tissues or cell types, at different developmental stages, or in response to different environmental or physiological conditions or inducer molecules. A promoter that drives gene expression in most cell types most of the time is commonly referred to as a "constitutive promoter." A promoter that drives gene expression in specific cell and tissue types is commonly referred to as a "cell-specific promoter" or "tissue-specific promoter," respectively. Promoters that cause expression of a gene at a specific stage of development or cell differentiation are commonly referred to as "development-specific promoters" or "cell differentiation-specific promoters." Promoters that induce and result in expression of a gene after cells are exposed to or treated with a promoter-inducing drug, biomolecule, chemical, ligand, light, etc. are commonly referred to as "inducible promoters" or "regulatable promoters." It is further recognized that in some embodiments, DNA fragments of different lengths have the same promoter activity, since the exact boundaries of regulatory sequences have not been completely defined in most cases.

[0147] The term "expression," as used herein, refers to the process by which a nucleic acid sequence or polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Collectively, the transcript and the encoded polypeptide may be referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0148] As used herein, "operably linked," "operable linkage," "operatively linked," or their grammatical equivalents refer to an arrangement of genetic elements, such as a promoter, enhancer, polyadenylation sequence, etc., such that operation (e.g., movement or activation) of a first genetic element has some effect on a second genetic element. The effect on the second genetic element may, but need not, be of the same type as the operation of the first genetic element. For example, two genetic elements are operably linked if movement of the first element causes activation of the second element. The effect on the second element may, but need not, be of the same type as the operation of the first element. For example, two elements are operably linked if movement of the first element causes activation of the second element. For example, a regulatory element, which may include a promoter sequence and / or an enhancer sequence, is operably linked to a coding region if the regulatory element helps to initiate transcription of the coding sequence. There may be intervening residues between the regulatory elements and the coding region so long as this functional relationship is maintained.

[0149] As used herein, "vector" refers to a macromolecule or association of macromolecules that contains or is associated with a polynucleotide and can be used to mediate delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. A vector generally contains genetic elements, such as regulatory elements, operably linked to a gene to facilitate expression of the gene in a target.

[0150] As used herein, "expression cassette" and "nucleic acid cassette" are used interchangeably to refer to a combination of nucleic acid sequences or elements that are expressed together or operably linked for expression. In some cases, an expression cassette refers to a combination of a gene or genes with regulatory elements that are operably linked for expression.

[0151] A "functional fragment" of a DNA or protein sequence refers to a fragment that retains a biological activity (either functional or structural) substantially similar to that of the full-length DNA or protein sequence. The biological activity of a DNA sequence may be the ability to affect expression in a manner attributable to the full-length sequence.

[0152] The terms "engineered," "synthetic," and "artificial" are used interchangeably herein to refer to entities modified by human intervention. For example, the terms can refer to non-naturally occurring polynucleotides or polypeptides. Engineered peptides can, but need not, have low sequence identity to naturally occurring human proteins (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity). For example, the VPR domain and the VP64 domain are synthetic transactivation domains. By non-limiting example, a nucleic acid may be modified by changing its sequence to one that does not occur in nature; a nucleic acid may be modified by ligating to a nucleic acid with which it is not naturally associated such that the ligated product has a function not present in the original nucleic acid; an engineered nucleic acid may be synthesized in vitro using a sequence that does not occur in nature; a protein may be modified by changing its amino acid sequence to a sequence that does not occur in nature; an engineered protein may acquire a new function or property. An "engineered" system contains at least one engineered component.

[0153] The term "tracrRNA" or "tracr sequence" refers to a CRISPR RNA that transactivates. The tracrRNA interacts with the CRISPR (cr) RNA to form a guide nucleic acid (e.g., guide RNA or gRNA) that can hybridize to a target nucleic acid and thereby direct an associated nuclease to the target nucleic acid. When the tracrRNA is engineered, it can 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., a tracrRNA of S. pyogenes, S. aureus, etc., or SEQ ID NO: **). tracrRNA can also refer to modified forms of tracrRNA that can contain nucleotide changes, such as deletions, insertions, or substitutions, variants, mutations, or chimeras. A tracrRNA may refer to a nucleic acid that may be at least about 60% identical to a wild-type exemplary tracrRNA (e.g., a tracrRNA from S. pyogenes, S. aureus, etc.) sequence over a stretch of at least six contiguous nucleotides. For example, a tracrRNA sequence may 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 (e.g., a tracrRNA from S. pyogenes, S. aureus, etc.) sequence over a section of at least six contiguous nucleotides. Type II tracrRNA sequences can be predicted on a genomic sequence by identifying regions that have complementarity to portions of repeat sequences in adjacent CRISPR arrays.

[0154] As used herein, "guide nucleic acid" or "guide polynucleotide" refers to a nucleic acid that can hybridize to a target nucleic acid, thereby directing an associated nuclease to the target nucleic acid. A guide nucleic acid can be RNA (guide RNA or gRNA). A guide nucleic acid can be DNA. A guide nucleic acid can be a mixture of RNA and DNA. A guide nucleic acid can include crRNA or tracrRNA, or a combination of both. A guide nucleic acid can be engineered. A guide nucleic acid can be programmed to specifically bind to a target nucleic acid. A portion of a target nucleic acid can be complementary to a portion of a guide nucleic acid. A strand of a double-stranded target polynucleotide that is complementary to and hybridizes with a guide nucleic acid can be referred to as the complementary strand. A strand of a double-stranded target polynucleotide that is complementary to the complementary strand and therefore not complementary to the guide nucleic acid can be referred to as the non-complementary strand. A guide nucleic acid can comprise a polynucleotide strand and can be referred to as a "single guide nucleic acid." A guide nucleic acid can comprise two polynucleotide strands and can be referred to as a "dual guide nucleic acid." Otherwise, the term "guide nucleic acid" may be generic, referring to both single and double guide nucleic acids. A guide nucleic acid may include a segment that may be referred to as a "nucleic acid targeting segment" or "nucleic acid targeting sequence" or "spacer." A nucleic acid targeting segment may include a subsegment that may be referred to as a "protein binding segment" or "protein binding sequence" or "Cas protein binding segment."

[0155] As used herein, the terms "gene editing" and "genome editing" may be used interchangeably. Gene editing or genome editing refers to changing the nucleic acid sequence of a gene or genome. Genome editing can include, for example, insertions, deletions, and mutations.

[0156] The term "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 identical or have a specified percentage of identical amino acid residues or nucleotides 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 using the BLOSUM62 scoring matrix setting parameters of word length (W) of 3, expectation (E) of 10, and presence of 11, gap cost at an extension of 1, and using a conditional composition score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of word length (W) of 2, expectation (E) of 1,000,000, and PAM30 scoring setting gap costs at 9 for open gaps and 1 for extended gaps for sequences shorter than 30 residues (default parameters for BLASTP are available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW using the Smith-Waterman homology search algorithm with parameters of match of 2, mismatch of -1, and gap of -1; MUSCLE using default parameters; MAFFT using parameters retree of 2 and maximum iterations of 1,000; Novafold using default parameters; and HMMER hmmalign using default parameters.

[0157] Variants of any of the enzymes described herein having one or more conservative amino acid substitutions are included in the present disclosure. Such conservative substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions can be achieved by substituting amino acids with similar hydrophobicity, polarity, and R chain length. Additionally or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions can be identified by identifying amino acid residues that are not mutated between species (e.g., residues that are not conserved without altering the basic function of the encoded protein). Such conservatively substituted variants can include variants having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of the systems described herein (e.g., the MG36 or MG39 systems described herein). In some embodiments, such conservatively substituted variants are functional variants. Such functional variants can include sequences with substitutions of key active site residues of the endonuclease such that the activity of the residues is not disrupted. In some embodiments, a functional variant of any of the systems described herein lacks a substitution of at least one of the conserved or functional residues called out in Figures 4 and 5. In some embodiments, a functional variant of any of the systems described herein lacks a substitution of all of the conserved or functional residues called out in Figures 4 and 5.

[0158] Conservative substitution tables providing functionally similar amino acids are available from various references (e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2002)). nd Edition (December 1993). The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine ​​(C), methionine (M).

[0159] As used herein, the term "RuvC_III domain" refers to the third, non-contiguous segment of the RuvC endonuclease domain (the RuvC nuclease domain is composed of three non-contiguous segments, RuvC_I, RuvC_II, and RuvC_III). RuvC domains or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or comparison to hidden Markov models (HMMs) constructed based on documented domain sequences (e.g., Pfam HMM PF18541 for RuvC_III).

[0160] 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 alignment to documented domain sequences, structural alignment to proteins with annotated domains, or comparison to hidden Markov models (HMMs) constructed based on documented domain sequences (e.g., Pfam HMM PF01844 for domain HNH).

[0161] As used herein, the term "recombinase" refers to an enzyme that mediates the recombination of DNA fragments located between recombinase recognition sequences, resulting in excision, insertion, inversion, exchange, or transposition of the DNA fragments located between the recombinase recognition sequences.

[0162] As used herein, the terms "recombining" or "recombination" in the context of nucleic acid modification (e.g., genomic modification) refer to a process in which two or more nucleic acid molecules, or two or more regions of a single nucleic acid molecule, are modified by the action of a recombinase protein. Recombination can result in, among other things, excision, insertion, inversion, exchange, or rearrangement of nucleic acid sequences within or between one or more nucleic acid molecules.

[0163] As used herein, the term "transposon" or "transposable element" refers to a nucleic acid sequence in a genome that is a mobile genetic element that can change its position in the genome. In some cases, the transposon transports additional "cargo DNA" that is excised from the genome. Transposons include, for example, retrotransposons, DNA transposons, autonomous and non-autonomous transposons, and class III transposons. The transposon nucleic acid sequence includes, for example, a gene encoding a cognate transposase, one or more recognition sequences for the transposase, or a combination thereof. In some cases, these transposons may differ by the type of nucleic acid they transpose, the type of repeats at the ends of the transposon, the type of cargo they carry, or the mode of transposition (i.e., self-repair or host-repair). As used herein, the term "transposase" or "transposases" refers to an enzyme that binds to the recognition sequence of the transposon and catalyzes its movement to another part of the genome. In some cases, movement may be by a cut-and-paste mechanism or replicative transposition.

[0164] As used herein, the term "Tn7" or "Tn7-like transposase" refers to a family of transposases that contain three major components: a heteromeric transposase (TnsA and / or TnsB) along with a regulatory protein (TnsC). In addition to the TnsABC transposition proteins, Tn7 elements can encode dedicated target site selection proteins, TnsD and TnsE. In conjunction with TnsABC, the sequence-specific DNA-binding protein TnsD directs transposition into a conserved site termed 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 into degradation sites of plasmids.

[0165] As used herein, the term "complex" refers to the joining of at least two components. Each of the two components may retain the properties / activities it had prior to forming the complex. The joining may be by covalent bonding, non-covalent bonding (i.e., hydrogen bonding, ionic interactions, van der Waals interactions, and hydrophobic bonding), the use of 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 may include a Cas protein and a guide nucleic acid.

[0166] In some cases, the CAST systems described herein include one or more Tn7 or Tn7-like transposases. In certain exemplary embodiments, the Tn7 or Tn7-like transposase comprises a multimeric protein complex. In certain exemplary embodiments, the multimeric protein complex includes TnsA, TnsB, TnsC, or TniQ. In these combinations, the transposases (TnsA, TnsB, TnsC, TniQ) may form a complex or fusion protein with each other.

[0167] As used herein, the terms "gene editing" and "genome editing" may be used interchangeably. Gene editing or genome editing refers to changing the nucleic acid sequence of a gene or genome. Genome editing can include, for example, insertions, deletions, and mutations.

[0168] As used herein, the term "Cas12k" (alternatively, "Class 2 VK type") refers to a subtype of V-type CRISPR system that has been found to be defective in nuclease activity (e.g., they may contain at least one defective RuvC domain that lacks at least one catalytic residue important for DNA cleavage). Such effector subtypes are generally associated with CAST systems.

[0169] As used herein, the term "IF-type" (alternatively, Class I IF-type CRISPR) refers to a subtype of Class I Type I CRISPR system. Such systems generally comprise a multi-component CRISPR effector including Cas8, Cas7, and Cas6 proteins. In some cases, such systems are found associated with CAST systems. In some cases, IF-type CRISPR systems comprise a crRNA that includes an 8-nt 5' handle for Cas8 and / or Cas5 binding, a 32-nt spacer bounded by six copies of Cas7 for target recognition, or a 20-nt 3' hairpin for Cas6 binding and pre-crRNA processing. In some cases, F-type systems utilize a 5'-CC PAM on the non-target strand for target binding.

[0170] As used herein, the term "functional domain (FD)" refers to a small protein that can facilitate protein interaction with DNA. Types of functional domains include, but are not limited to, DNA-binding domains (DBDs) and chromatin modulating domains (CMDs). Non-limiting examples of functional domains include human histone 1 central globular domain (H1core), high mobility group nucleosome binding domain 1 (HMGN1), chromobox 5 (Cbx5), and Saccharolobus solfataricus sso7d. In some embodiments, the functional domains described herein can be included in fusion proteins with the systems or components thereof described herein. In some embodiments, the fusion proteins can exhibit increased activity in cells compared to the non-fused protein.

[0171] In accordance with IUPAC convention, the following abbreviations are used throughout the examples: A=adenine C=cytosine G = guanine T=thymine R = adenine or guanine Y = cytosine or thymine S = guanine or cytosine W = adenine or thymine K = guanine or thymine M = adenine or cytosine B=C, G, or T D=A, G, or T H=A, C, or T V=A, C, or G

[0172] overview The discovery of new Cas enzymes with unique functionality and structure could further disrupt deoxyribonucleic acid (DNA) editing technology, offering the potential to improve speed, specificity, functionality, and ease of use. Compared to the predicted prevalence of clustered regularly interspaced short palindromic repeats (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 due to the inability to easily cultivate vast numbers of microbial species under laboratory conditions. Metagenomic sequencing from natural environmental niches representing numerous microbial species could dramatically increase the number of documented new CRISPR / Cas systems and potentially expedite the discovery of novel oligonucleotide editing functions. A recent example of the fruitfulness of such an approach is demonstrated by the 2016 discovery of the CasX / CasY CRISPR system from metagenomic analysis of natural microbial communities.

[0173] CRISPR / Cas systems are RNA-directed nuclease complexes that have been described to function as adaptive immune systems in microorganisms. In their natural context, CRISPR / Cas systems occur in CRISPR (clustered regularly interspaced short palindromic repeats) operons or loci, which generally contain two parts: (i) an array of short repeat sequences (30-40 bp) separated by equally short spacer sequences that encode RNA-based targeting elements; and (ii) an ORF encoding a Cas, which encodes a nuclease polypeptide directed by the RNA-based targeting element flanked by accessory proteins / enzymes. 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 (PAM is typically a sequence not commonly represented in the host genome). Depending on the exact function and organization of the system, CRISPR-Cas systems are commonly organized into two classes, five types, and 16 subtypes based on shared functional characteristics and evolutionary similarities (see Figure 1).

[0174] Class 1 CRISPR-Cas systems have large multi-subunit effector complexes and include types I, III, and IV.

[0175] Type I CRISPR-Cas systems are considered to be of intermediate complexity in terms of components. In Type I CRISPR-Cas systems, an array of RNA targeting elements is transcribed as a long precursor crRNA (pre-crRNA) that is processed at the repeat element, releasing a short mature crRNA that directs the nuclease complex to the nucleic acid target, followed by a suitable short consensus sequence called a protospacer adjacent motif (PAM). This processing occurs via the endoribonuclease subunit (Cas6) of a larger endonuclease complex called Cascade, which also contains the nuclease (Cas3) protein component of the crRNA-directed nuclease complex. Cas I nuclease functions primarily as a DNA nuclease.

[0176] Type III CRISPR systems can be characterized by the presence of a central nuclease known as Cas10, along with repeat-associated mysterious proteins (RAMPs) containing Csm or Cmr protein subunits. Similar to type I systems, mature crRNA is processed from pre-crRNA using a Cas6-like enzyme. Unlike type I and II systems, type III systems are thought to target and cleave DNA-RNA duplexes (such as the DNA strand used as a template for RNA polymerase).

[0177] Type IV CRISPR-Cas systems possess an effector complex containing a highly reduced large subunit nuclease (csf1), two genes for RAMP proteins of the Cas5 (csf3) and Cas7 (csf2) family, and in some cases a predicted small subunit gene; such systems are typically found on endogenous plasmids.

[0178] Class 2 CRISPR-Cas systems generally have a single polypeptide multi-domain nuclease effector and include types II, V, and VI.

[0179] Type II CRISPR-Cas systems are considered the simplest in terms of components. In type II CRISPR-Cas systems, processing the CRISPR array into mature crRNA does not require the presence of a specialized endonuclease subunit, but rather a small transcoding crRNA (tracrRNA) with a region complementary to the array repeat sequence. The tracrRNA interacts with both its corresponding effector nuclease (e.g., Cas9) and the repeat sequence to form a precursor dsRNA structure, which is cleaved by endogenous RNAse III to generate the mature effector enzyme loaded with both the tracrRNA and crRNA. Cas II nucleases are documented as DNA nucleases. Type II effectors generally exhibit a structure containing a RuvC-like endonuclease domain that fits into an RNase H fold with an unrelated HNH nuclease domain inserted into the RuvC-like nuclease domain fold. The RuvC-like domain is responsible for cleavage of the target (e.g., crRNA-complementary) DNA strand, while the HNH domain is responsible for cleavage of the displacement DNA strand.

[0180] Type V CRISPR-Cas systems are characterized by a nuclease effector (e.g., Cas12) structure similar to that of type II effectors, containing a RuvC-like domain. Like type II, most (but not all) type V CRISPR systems use a tracrRNA to process pre-crRNA into mature crRNA. However, unlike type II systems, which require RNAse III to cleave the pre-crRNA into multiple crRNAs, type V systems can cleave the pre-crRNA using the effector nuclease itself. Like type II CRISPR-Cas systems, type V CRISPR-Cas systems are also documented as DNA nucleases. Unlike type II CRISPR-Cas systems, some type V enzymes (e.g., Cas12a) appear to have robust single-strand nonspecific deoxyribonuclease activity that is activated by the first crRNA-directed cleavage of the double-stranded target sequence.

[0181] Type VI CRIPSR-Cas systems possess an RNA-guided RNA endonuclease. Instead of a RuvC-like domain, the single polypeptide effector of type VI systems (e.g., Cas13) contains two HEPN ribonuclease domains. Unlike both type II and type V systems, in some cases, type VI systems also appear to not require a tracrRNA to process pre-crRNA into crRNA. However, like type V systems, some type VI systems (e.g., C2C2) appear to possess robust single-strand nonspecific nuclease (ribonuclease) activity that is activated by the first crRNA-directed cleavage of the target RNA.

[0182] Due to their simpler structure, Class 2 CRISPR-Cas have been most widely adopted for engineering and development as engineered nuclease / genome editing applications.

[0183] One of the initial adaptations of such a system for in vivo use involved (i) purified recombinantly expressed full-length Cas9 (e.g., a class 2, type II Cas enzyme) isolated from S. pyogenes SF370, (ii) purified mature, approximately 42-nt crRNA (total crRNA transcribed in vitro from a synthetic DNA template carrying a T7 promoter sequence) carrying an approximately 20-nt 5' sequence complementary to the target DNA sequence desired to be cleaved, followed by a 3' tracr binding sequence, (iii) purified tracrRNA transcribed in vitro from a synthetic DNA template carrying a T7 promoter sequence, and (iv) Mg 2+ Subsequent improved, engineered systems involved (ii) crRNA joined to the 5' end of (iii) by a linker (e.g., GAAA) to form a single fusion synthetic guide RNA (sgRNA) that could itself guide Cas9 to the target (compare the top and bottom panels of Figure 2).

[0184] Such engineered systems can be adapted for use in 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 attached to a 3' tracr binding sequence, a linker, and the tracrRNA sequence) under a suitable polymerase III promoter (e.g., a U6 promoter).

[0185] Transposons are mobile elements that can move between locations within a genome. Such transposons have evolved to limit their adverse effects on the host. Various regulatory mechanisms are used to maintain translocation at low frequency and sometimes coordinate translocation with various cellular processes. Some prokaryotic transposons can also combine functions that benefit the host or otherwise help maintain the element. Certain transposons may also have evolved strict regulatory mechanisms for target site selection, the most prominent example being the Tn7 family.

[0186] Transposon Tn7 and similar elements are reservoirs for antibiotic resistance and pathogenic functions in clinical settings and may encode other adaptive functions in natural environments. The Tn7 system has evolved mechanisms to maximize element dispersal, for example, by almost completely avoiding integration into critical host genes but by recognizing mobile plasmids and bacteriophages that can transfer Tn7 between host bacteria.

[0187] Tn7 and Tn7-like elements control where and when they insert, and may possess one pathway that directs insertion into a single, conserved location within the bacterial genome, and a second pathway that appears to be adapted to maximize targeting onto mobile plasmids that can transport elements between bacteria (see Figure 3). The link between Tn7-like transposons and CRISPR-Cas systems suggests that transposons may have hijacked CRISPR effectors that generate R-loops at target sites, facilitating transposon spread through plasmids and phages.

[0188] Functional domain fusion The lack of activity of some enzyme effectors in human cells may be due to inaccessibility of the target site, for example, due to strict chromatin structure. Therefore, fusing functional domains to the effector can improve enzyme activity. For example, fusing Taq polymerase to the sso7d (ds) DNA-binding protein improved enzyme processivity, requiring much less enzyme and a much shorter extension time (Wang et al., 2004). Similarly, the excision-editing efficiency of CjCas9 in K562 cells improved when fused with various functional domains (Ding et al., 2019).

[0189] MG36 series In some embodiments, provided herein is an MG36 system for translocating a cargo nucleotide sequence into a target nucleic acid site. See 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 a Class 2 Type II Cas effector and at least one engineered guide polynucleotide configured to hybridize to the target nucleic acid site. In some embodiments, the Class 2 Type II Cas effector comprises a RuvC domain and an 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.

[0190] In some cases, the cargo nucleotide sequence is adjacent to a left-hand transposase recognition sequence. In some cases, the cargo nucleotide sequence is adjacent to a right-hand transposase recognition sequence. In some cases, the cargo nucleotide sequence is adjacent to a left-hand transposase recognition sequence and a right-hand transposase recognition sequence.

[0191] In some cases, the target nucleic acid comprises a target nucleic acid site. In some cases, the target nucleic acid comprises a PAM sequence that is compatible with a 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. In some cases, the PAM sequence is located 5' of the target nucleic acid site.

[0192] In some cases, the engineered guide polynucleotide is configured to bind to a Class 2 Type II Cas endonuclease. In some cases, the Class 2 Type II Cas effector comprises 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 Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least about 70% identity to SEQ ID NO: 1. In some cases, a Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least about 75% identity to SEQ ID NO:1. In some cases, a Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least about 80% identity to SEQ ID NO:1. In some cases, a Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least about 85% identity to SEQ ID NO:1. In some cases, a Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least about 90% identity to SEQ ID NO:1. In some cases, a Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least about 91% identity to SEQ ID NO:1. In some cases, a Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least about 92% identity to SEQ ID NO:1. In some cases, a Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least about 93% identity to SEQ ID NO:1. In some cases, the Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least about 94% identity to SEQ ID NO:1.In some cases, a Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least about 95% identity to SEQ ID NO:1. In some cases, a Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least about 96% identity to SEQ ID NO:1. In some cases, a Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least about 97% identity to SEQ ID NO:1. In some cases, a Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least about 98% identity to SEQ ID NO:1. In some cases, a Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having at least about 99% identity to SEQ ID NO:1. In some cases, a Class 2 Type II Cas effector comprises a polypeptide comprising a sequence having 100% identity to SEQ ID NO:1.

[0193] In some cases, the recombinase or transposase complex comprises at least one polypeptide (e.g., at least one, two, three, four, five, six, or more than six 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises at least one polypeptide comprising a sequence having 100% identity to any one of SEQ ID NOs: 2-5.

[0194] In some cases, a 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, a 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 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 80% 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 85% 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 90% 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 91% 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 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 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 96% 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 97% 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 98% 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 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 100% identity to any one of SEQ ID NOs: 2-5.

[0195] In some embodiments, the systems disclosed herein comprise at least one engineered guide polynucleotide, e.g., a gRNA.

[0196] In some embodiments, provided herein are engineered guide polynucleotides, such as guide RNAs (gRNAs).

[0197] In some cases, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 contiguous nucleotides that has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to 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 70% 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 75% identical to SEQ ID NO: 11. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 60-80 contiguous nucleotides that are at least about 80% relative to SEQ ID NO:11. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 60-80 contiguous nucleotides that are at least about 85% relative to SEQ ID NO:11. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 60-80 contiguous nucleotides that are at least about 90% relative to SEQ ID NO:11. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 60-80 contiguous nucleotides that are at least about 91% relative to SEQ ID NO:11. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 60-80 contiguous nucleotides that are at least about 92% relative to SEQ ID NO:11. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 60-80 contiguous nucleotides that are at least about 93% relative to SEQ ID NO:11.In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 60-80 contiguous 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 contiguous 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 contiguous 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 contiguous 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 contiguous 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 are 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 are 100% identical to SEQ ID NO:11.

[0198] In some embodiments, the guide RNA comprises various structural elements, including, but not limited to, a spacer sequence that binds to a protospacer sequence (target sequence), a crRNA, and an optional tracrRNA. In some embodiments, the guide RNA comprises a crRNA that comprises a spacer sequence. In some embodiments, the guide RNA additionally comprises a tracrRNA or a modified tracrRNA.

[0199] In some embodiments, the systems provided herein include 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 the target sequence. For example, the crRNA is part of, or considered part of, the guide RNA, or is included in the guide RNA, e.g., a crRNA:tracrRNA chimera.

[0200] In some embodiments, the guide RNA comprises synthetic or modified nucleotides. In some embodiments, the guide RNA comprises one or more internucleoside linkers modified from natural phosphodiester. In some embodiments, the internucleoside linker of the guide RNA, or all of its contiguous nucleotide sequence, is modified. For example, in some embodiments, the internucleoside linkage comprises sulfur (S), such as a phosphorothioate internucleoside linkage.

[0201] In some embodiments, the guide RNA comprises a modification to the ribose sugar or nucleobase. In some embodiments, the guide RNA comprises one or more nucleosides comprising a modified sugar moiety, where the modified sugar moiety is a modification of the sugar moiety compared to the ribose sugar moiety found in deoxyribose nucleic acids (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, substitution with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g., locked nucleic acids (LNA)), or an unlinked ribose ring, which typically lacks a bond between the C2 and C3 carbons (e.g., UNA). In some embodiments, the sugar-modified nucleoside comprises a bicyclohexose nucleic acid or a tricyclic nucleic acid. In some embodiments, the modified nucleoside comprises a nucleoside in which the sugar moiety is replaced with a non-sugar moiety, such as a peptide nucleic acid (PNA) or morpholino nucleic acid.

[0202] In some embodiments, the guide RNA comprises one or more modified sugars. In some embodiments, sugar modifications include modifications made by changing the substituent on the ribose ring to a group other than hydrogen or to the 2'-OH group naturally found in DNA and RNA nucleosides. In some embodiments, the substituent is introduced at the 2', 3', 4', or 5' position, or a combination thereof. In some embodiments, nucleosides having modified sugar moieties include 2'-modified nucleosides, e.g., 2'-substituted nucleosides. 2'-sugar-modified nucleosides, in some embodiments, are nucleosides having a substituent other than -H or -OH at the 2' position (2'-substituted nucleosides) or include a 2'-linked biradical, and include 2'-substituted nucleosides and LNA (2'-4' biradical 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).

[0203] In some embodiments, the guide RNA comprises one or more modified sugars. In some embodiments, the guide RNA comprises only modified sugars. In certain embodiments, the guide RNA comprises more than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2'-O-methoxyethyl group. In some embodiments, the guide RNA comprises both an internucleoside linker modification and a nucleoside modification.

[0204] In some cases, the guide RNA comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a fungal genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a plant genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a human genomic polynucleotide sequence.

[0205] 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 RNAs are 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 50 The length is about 120, about 50 to about 140, about 50 to about 160, about 50 to about 180, about 50 to about 200, about 50 to about 220, about 50 to about 240, about 100 to about 120, about 100 to about 140, about 100 to about 160, about 100 to about 180, about 100 to about 200, about 100 to about 220, about 100 to about 240, about 160 to about 180, about 160 to about 200, about 160 to about 220, or about 160 to about 240 nucleotides.

[0206] In some embodiments, the left-hand 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-hand 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-hand 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-hand recombinase sequence comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 17-18. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 17-18. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 17-18. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 17-18. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 17-18. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 17-18. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 17-18. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 17-18.In some cases, the left-hand recombinase sequence comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 17-18. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 17-18. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 17-18. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 17-18. In some cases, the left-hand recombinase sequence comprises a sequence having 100% identity to any one of SEQ ID NOs: 17-18.

[0207] 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 comprises a sequence having at least about 85% identity to SEQ ID NO:19. In some cases, the right recombinase sequence comprises a sequence having at least about 90% identity to SEQ ID NO:19. In some cases, the right recombinase sequence comprises a sequence having at least about 91% identity to SEQ ID NO:19. In some cases, the right recombinase sequence comprises a sequence having at least about 92% identity to SEQ ID NO:19. In some cases, the right recombinase sequence comprises a sequence having at least about 93% identity to SEQ ID NO:19. In some cases, the right recombinase sequence comprises a sequence having at least about 94% identity to SEQ ID NO:19. In some cases, the right recombinase sequence comprises a sequence having at least about 95% identity to SEQ ID NO:19. In some cases, the right recombinase sequence comprises a sequence having at least about 96% identity to SEQ ID NO: 19. In some cases, the right recombinase sequence comprises 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.

[0208] 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.

[0209] MG39 series In some embodiments, provided herein is an MG39 system for transposing a cargo nucleotide sequence into a target nucleic acid site. With reference to Figures 5A and 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 a class 2 V-type Cas effector and an engineered guide polynucleotide configured to hybridize to a target nucleotide sequence. In some embodiments, the class 2 V-type Cas effector comprises a RuvC domain. In some embodiments, the system comprises a Tn7-type transposase complex configured to bind to the Cas effector complex, wherein the Tn7-type transposase complex comprises a TnsA subunit.

[0210] In some cases, the cargo nucleotide sequence is adjacent to a left-hand transposase recognition sequence. In some cases, the cargo nucleotide sequence is adjacent to a right-hand transposase recognition sequence. In some cases, the cargo nucleotide sequence is adjacent to a left-hand transposase recognition sequence and a right-hand transposase recognition sequence.

[0211] In some cases, the target nucleic acid comprises a target nucleic acid site. In some cases, the target nucleic acid comprises a PAM sequence that is compatible with a 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. In some cases, the PAM sequence is located 5' of the target nucleic acid site.

[0212] In some cases, the engineered guide polynucleotide is configured to bind to a Class 2 type V Cas endonuclease. In some cases, the Class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 6. In some cases, the Class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 70% identity to SEQ ID NO: 6. In some cases, a Class 2 type-V Cas effector comprises a polypeptide comprising a sequence having at least about 75% identity to SEQ ID NO:6. In some cases, a Class 2 type-V Cas effector comprises a polypeptide comprising a sequence having at least about 80% identity to SEQ ID NO:6. In some cases, a Class 2 type-V Cas effector comprises a polypeptide comprising a sequence having at least about 85% identity to SEQ ID NO:6. In some cases, a Class 2 type-V Cas effector comprises a polypeptide comprising a sequence having at least about 90% identity to SEQ ID NO:6. In some cases, a Class 2 type-V Cas effector comprises a polypeptide comprising a sequence having at least about 91% identity to SEQ ID NO:6. In some cases, a Class 2 type-V Cas effector comprises a polypeptide comprising a sequence having at least about 92% identity to SEQ ID NO:6. In some cases, a Class 2 type-V Cas effector comprises a polypeptide comprising a sequence having at least about 93% identity to SEQ ID NO:6. In some cases, the Class 2 V-type Cas effector comprises a polypeptide comprising a sequence having at least about 94% identity to SEQ ID NO:6.In some cases, a Class 2 type-V Cas effector comprises a polypeptide comprising a sequence having at least about 95% identity to SEQ ID NO:6. In some cases, a Class 2 type-V Cas effector comprises a polypeptide comprising a sequence having at least about 96% identity to SEQ ID NO:6. In some cases, a Class 2 type-V Cas effector comprises a polypeptide comprising a sequence having at least about 97% identity to SEQ ID NO:6. In some cases, a Class 2 type-V Cas effector comprises a polypeptide comprising a sequence having at least about 98% identity to SEQ ID NO:6. In some cases, a Class 2 type-V Cas effector comprises a polypeptide comprising a sequence having at least about 99% identity to SEQ ID NO:6. In some cases, a Class 2 type-V Cas effector comprises a polypeptide comprising a sequence having 100% identity to SEQ ID NO:6.

[0213] In some cases, the Tn7-type transposase complex comprises at least one polypeptide (e.g., at least one, two, three, four, five, six, or more than six 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:8-10. In some cases, the Tn7-type transposase complex comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises at least one polypeptide comprising a sequence having 100% identity to any one of SEQ ID NOs: 8-10.

[0214] In some cases, a Tn7-type 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: 8-10. In some cases, a 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: 8-10. In some cases, a 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, a 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, a 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, a 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, a 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: 8-10. In some cases, a 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: 8-10. In some cases, a 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: 8-10. In some cases, a 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, a 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, a 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, a 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, a 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 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:8-10.

[0215] In some cases, a Tn7-type transposase complex comprises a TnsA component 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 NO: 7. In some cases, a Tn7-type transposase complex comprises a TnsA component comprising a sequence having at least about 70% identity to SEQ ID NO:7. In some cases, a Tn7-type transposase complex comprises a TnsA component comprising a sequence having at least about 75% identity to SEQ ID NO:7. In some cases, a Tn7-type transposase complex comprises a TnsA component comprising a sequence having at least about 80% identity to SEQ ID NO:7. In some cases, a Tn7-type transposase complex comprises a TnsA component comprising a sequence having at least about 85% identity to SEQ ID NO:7. In some cases, a Tn7-type transposase complex comprises a TnsA component comprising a sequence having at least about 90% identity to SEQ ID NO:7. In some cases, a Tn7-type transposase complex comprises a TnsA component comprising a sequence having at least about 91% identity to SEQ ID NO:7. In some cases, a Tn7-type transposase complex comprises a TnsA component comprising a sequence having at least about 92% identity to SEQ ID NO:7. In some cases, a Tn7-type transposase complex comprises 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 comprises 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 comprises a TnsA component comprising a sequence having at least about 95% identity to SEQ ID NO: 7.In some cases, a Tn7-type transposase complex comprises a TnsA component comprising a sequence having at least about 96% identity to SEQ ID NO:7. In some cases, a Tn7-type transposase complex comprises a TnsA component comprising a sequence having at least about 97% identity to SEQ ID NO:7. In some cases, a Tn7-type transposase complex comprises a TnsA component comprising a sequence having at least about 98% identity to SEQ ID NO:7. In some cases, a Tn7-type transposase complex comprises a TnsA component comprising a sequence having at least about 99% identity to SEQ ID NO:7. In some cases, a Tn7-type transposase complex comprises a TnsA component comprising a sequence having 100% identity to SEQ ID NO:7.

[0216] In some cases, the Tn7-type transposase complex comprises a TnsB component 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 NO: 8. In some cases, the Tn7-type transposase complex comprises a TnsB component comprising a sequence having at least about 70% identity to SEQ ID NO:8. In some cases, a Tn7-type transposase complex comprises a TnsB component comprising a sequence having at least about 75% identity to SEQ ID NO:8. In some cases, a Tn7-type transposase complex comprises a TnsB component comprising a sequence having at least about 80% identity to SEQ ID NO:8. In some cases, a Tn7-type transposase complex comprises a TnsB component comprising a sequence having at least about 85% identity to SEQ ID NO:8. In some cases, a Tn7-type transposase complex comprises a TnsB component comprising a sequence having at least about 90% identity to SEQ ID NO:8. In some cases, a Tn7-type transposase complex comprises a TnsB component comprising a sequence having at least about 91% identity to SEQ ID NO:8. In some cases, a Tn7-type transposase complex comprises a TnsB component comprising a sequence having at least about 92% identity to SEQ ID NO:8. In some cases, a Tn7-type transposase complex comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises a TnsB component comprising a sequence having 100% identity to SEQ ID NO:8.

[0217] In some cases, a Tn7-type transposase complex comprises a TnsC component 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 NO: 9. In some cases, a Tn7-type transposase complex comprises a TnsC component comprising a sequence having at least about 70% identity to SEQ ID NO:9. In some cases, a Tn7-type transposase complex comprises a TnsC component comprising a sequence having at least about 75% identity to SEQ ID NO:9. In some cases, a Tn7-type transposase complex comprises a TnsC component comprising a sequence having at least about 80% identity to SEQ ID NO:9. In some cases, a Tn7-type transposase complex comprises a TnsC component comprising a sequence having at least about 85% identity to SEQ ID NO:9. In some cases, a Tn7-type transposase complex comprises a TnsC component comprising a sequence having at least about 90% identity to SEQ ID NO:9. In some cases, a Tn7-type transposase complex comprises a TnsC component comprising a sequence having at least about 91% identity to SEQ ID NO:9. In some cases, a Tn7-type transposase complex comprises a TnsC component comprising a sequence having at least about 92% identity to SEQ ID NO:9. In some cases, a Tn7-type transposase complex comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises a TnsC component comprising a sequence having 100% identity to SEQ ID NO:9.

[0218] In some cases, the Tn7-type transposase complex comprises a TniQ 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: 10. In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having at least about 70% identity to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having at least about 75% identity to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having at least about 80% identity to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having at least about 85% identity to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having at least about 90% identity to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having at least about 91% identity to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having at least about 92% identity to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having at least about 93% identity to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having at least about 94% identity to SEQ ID NO: 10.In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having at least about 95% identity to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having at least about 96% identity to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having at least about 97% identity to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having at least about 98% identity to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having at least about 99% identity to SEQ ID NO: 10. In some cases, the Tn7-type transposase complex comprises a TniQ polypeptide comprising a sequence having 100% identity to SEQ ID NO: 10.

[0219] In some embodiments, the systems disclosed herein comprise at least one engineered guide polynucleotide, e.g., a gRNA.

[0220] In some embodiments, provided herein are engineered guide polynucleotides, such as guide RNAs (gRNAs).

[0221] In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides that has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 contiguous nucleotides that is at least about 70% identical to any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 75% of any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 80% of any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 85% of any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 90% of any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 91% of any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 contiguous nucleotides that are at least about 92% of any one of SEQ ID NOs: 13-16.In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 93% of any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 94% of any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 95% of any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 96% of any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 97% of any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 98% of any one of SEQ ID NOs: 13-16. In some embodiments, the engineered guide polynucleotide comprises a sequence comprising at least 46-80 contiguous nucleotides that are 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 contiguous nucleotides that are 100% identical to any one of SEQ ID NOs: 13-16.

[0222] In some embodiments, the guide RNA comprises various structural elements, including, but not limited to, a spacer sequence that binds to a protospacer sequence (target sequence), a crRNA, and an optional tracrRNA. In some embodiments, the guide RNA comprises a crRNA that comprises a spacer sequence. In some embodiments, the guide RNA additionally comprises a tracrRNA or a modified tracrRNA.

[0223] In some embodiments, the systems provided herein include 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 the target sequence. For example, the crRNA is part of, or considered part of, the guide RNA, or is included in the guide RNA, e.g., a crRNA:tracrRNA chimera.

[0224] In some embodiments, the guide RNA comprises synthetic or modified nucleotides. In some embodiments, the guide RNA comprises one or more internucleoside linkers modified from natural phosphodiester. In some embodiments, the internucleoside linker of the guide RNA, or all of its contiguous nucleotide sequence, is modified. For example, in some embodiments, the internucleoside linkage comprises sulfur (S), such as a phosphorothioate internucleoside linkage.

[0225] In some embodiments, the guide RNA comprises a modification to the ribose sugar or nucleobase. In some embodiments, the guide RNA comprises one or more nucleosides comprising a modified sugar moiety, where the modified sugar moiety is a modification of the sugar moiety compared to the ribose sugar moiety found in deoxyribose nucleic acids (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, substitution with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g., locked nucleic acids (LNA)), or an unlinked ribose ring, which typically lacks a bond between the C2 and C3 carbons (e.g., UNA). In some embodiments, the sugar-modified nucleoside comprises a bicyclohexose nucleic acid or a tricyclic nucleic acid. In some embodiments, the modified nucleoside comprises a nucleoside in which the sugar moiety is replaced with a non-sugar moiety, such as a peptide nucleic acid (PNA) or morpholino nucleic acid.

[0226] In some embodiments, the guide RNA comprises one or more modified sugars. In some embodiments, sugar modifications include modifications made by changing the substituent on the ribose ring to a group other than hydrogen or to the 2'-OH group naturally found in DNA and RNA nucleosides. In some embodiments, the substituent is introduced at the 2', 3', 4', or 5' position, or a combination thereof. In some embodiments, nucleosides having modified sugar moieties include 2'-modified nucleosides, e.g., 2'-substituted nucleosides. 2'-sugar-modified nucleosides, in some embodiments, are nucleosides having a substituent other than -H or -OH at the 2' position (2'-substituted nucleosides) or include a 2'-linked biradical, and include 2'-substituted nucleosides and LNA (2'-4' biradical 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).

[0227] In some embodiments, the guide RNA comprises one or more modified sugars. In some embodiments, the guide RNA comprises only modified sugars. In certain embodiments, the guide RNA comprises more than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2'-O-methoxyethyl group. In some embodiments, the guide RNA comprises both an internucleoside linker modification and a nucleoside modification.

[0228] In some cases, the guide RNA comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a fungal genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a plant genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a human genomic polynucleotide sequence.

[0229] 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 RNAs are 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 50 The length is about 120, about 50 to about 140, about 50 to about 160, about 50 to about 180, about 50 to about 200, about 50 to about 220, about 50 to about 240, about 100 to about 120, about 100 to about 140, about 100 to about 160, about 100 to about 180, about 100 to about 200, about 100 to about 220, about 100 to about 240, about 160 to about 180, about 160 to about 200, about 160 to about 220, or about 160 to about 240 nucleotides.

[0230] In some cases, the left-hand 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-hand recombinase sequence comprises a sequence having at least about 70% identity to SEQ ID NO: 20. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 75% identity to SEQ ID NO: 20. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 80% identity to SEQ ID NO:20. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 85% identity to SEQ ID NO:20. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 90% identity to SEQ ID NO:20. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 91% identity to SEQ ID NO:20. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 92% identity to SEQ ID NO:20. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 93% identity to SEQ ID NO:20. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 94% identity to SEQ ID NO:20. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 95% identity to SEQ ID NO:20. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 96% identity to SEQ ID NO: 20. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 97% identity to SEQ ID NO: 20. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 98% identity to SEQ ID NO: 20.In some cases, the left-hand recombinase sequence comprises a sequence having at least about 99% identity to SEQ ID NO: 20. In some cases, the left-hand recombinase sequence comprises a sequence having 100% identity to SEQ ID NO: 20.

[0231] 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 comprises a sequence having at least about 85% identity to SEQ ID NO:21. In some cases, the right recombinase sequence comprises a sequence having at least about 90% identity to SEQ ID NO:21. In some cases, the right recombinase sequence comprises a sequence having at least about 91% identity to SEQ ID NO:21. In some cases, the right recombinase sequence comprises a sequence having at least about 92% identity to SEQ ID NO:21. In some cases, the right recombinase sequence comprises a sequence having at least about 93% identity to SEQ ID NO:21. In some cases, the right recombinase sequence comprises a sequence having at least about 94% identity to SEQ ID NO:21. In some cases, the right recombinase sequence comprises a sequence having at least about 95% identity to SEQ ID NO:21. In some cases, the right recombinase sequence comprises a sequence having at least about 96% identity to SEQ ID NO: 21. In some cases, the right recombinase sequence comprises 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.

[0232] In some cases, the class 2 V-type Cas effector and Tn7-type transposase complex are encoded by a polynucleotide sequence comprising less than about 20 kilobases, less than about 15 kilobases, less than about 10 kilobases, or less than about 5 kilobases.

[0233] MG64 series In some embodiments, provided herein is an MG64 system for transposing a cargo nucleotide sequence into a target nucleic acid site. In some embodiments, the system comprises a double-stranded nucleic acid comprising the cargo nucleotide sequence. In some embodiments, the cargo nucleotide sequence is configured to interact with a Tn7-type or Tn5053-type transposase complex. In some embodiments, the system comprises a Cas effector complex. In some embodiments, the Cas effector complex comprises a class 2 V-type Cas effector and an engineered guide polynucleotide configured to hybridize to a target nucleotide sequence. In some embodiments, the system comprises a Tn7-type or Tn5053-type transposase complex configured to bind to the Cas effector complex. In some embodiments, the class 2 V-type Cas effector comprises a RuvC domain.

[0234] In some cases, the cargo nucleotide sequence is adjacent to a left-hand transposase recognition sequence. In some cases, the cargo nucleotide sequence is adjacent to a right-hand transposase recognition sequence. In some cases, the cargo nucleotide sequence is adjacent to a left-hand transposase recognition sequence and a right-hand transposase recognition sequence.

[0235] In some cases, the system further comprises a target nucleic acid comprising a target nucleic acid site. In some embodiments, the system further comprises a PAM sequence compatible with the nuclease adjacent to the target nucleic acid site. In some cases, the PAM sequence is located 3' of the target nucleic acid site. In some cases, the PAM sequence is located 5' of the target nucleic acid site. In some cases, the PAM sequence comprises 5'-nGTn-3' or 5'-nGTt-3'.

[0236] In some cases, the engineered guide polynucleotide is configured to bind to a Class 2 Type V Cas endonuclease. In some cases, the Class 2 Type V Cas effector comprises a polypeptide comprising a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, a Class 2 V-type 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. In some cases, a Class 2 V-type 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, a Class 2 V-type 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, a Class 2 V-type 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 V-type 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 V-type 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, a Class 2 V-type 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, a Class 2 V-type 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, a Class 2 V-type Cas effector comprises a polypeptide comprising a sequence having at least about 93% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147. In some cases, a Class 2 V-type 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, a Class 2 V-type 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, a Class 2 V-type 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, a Class 2 V-type 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, a Class 2 V-type Cas effector comprises 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, a Class 2 V-type Cas effector comprises 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 V-type Cas effector comprises a polypeptide comprising a sequence having 100% identity to any one of SEQ ID NOs: 22, 26, 30, 34, 55-89, 104, and 147.

[0237] In some cases, the Tn7-type transposase complex 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 80%, at least about 85 ... In some cases, the Tn7-type transposase complex comprises at least one polypeptide (e.g., at least one, two, three, four, five, six, or more than six polypeptides) comprising a sequence having 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises at least one polypeptide 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 comprises at least one polypeptide 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, a Tn7-type transposase complex comprises at least one polypeptide comprising a sequence having at least about 90% 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, a Tn7-type transposase complex comprises at least one polypeptide 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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 comprises at least one polypeptide 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, a Tn7-type transposase complex comprises at least one polypeptide comprising a sequence having at least about 97% 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, a Tn7-type transposase complex comprises at least one polypeptide 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, a Tn7-type transposase complex comprises at least one polypeptide 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises TnsB, TnsC, and TniQ.

[0238] In some cases, a Tn7-type 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, a 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, a 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, a 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, a 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150.In some cases, a 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, a 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, a 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, a 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, a 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, a 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150.In some cases, a 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, a 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, a 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. In some cases, a 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150. 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: 23-25, 27-29, 31-33, 35-37, 101-103, 105-107, and 148-150.

[0239] In some cases, the Tn7-type transposase complex comprises TnsB, TnsC, and TniQ polypeptides, each 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 a variant thereof. In some cases, the Tn7-type transposase complex comprises a TnsB polypeptide comprising a sequence substantially identical to any one of SEQ ID NO: 8 or a variant thereof. In some cases, the Tn7-type transposase complex comprises TnsB, TnsC, and TniQ polypeptides, each 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 a variant thereof.

[0240] In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides that has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 70% of 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 that comprises at least 46-80 contiguous nucleotides that are at least about 75% of 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 that comprises at least 46-80 contiguous nucleotides that are at least about 80% of 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 that comprises at least 46-80 contiguous nucleotides that are at least about 85% of 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 that comprises at least 46-80 contiguous nucleotides that are at least about 90% of 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 that comprises at least 46-80 contiguous nucleotides that are at least about 91% of 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 that comprises at least 46-80 contiguous nucleotides that are at least about 92% of 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 that comprises at least 46-80 contiguous nucleotides that are at least about 93% of 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 that comprises at least 46-80 contiguous nucleotides that are at least about 94% of 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 that comprises at least 46-80 contiguous nucleotides that are at least about 95% of 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 that comprises at least 46-80 contiguous nucleotides that are at least about 96% of 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 97% of 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 that comprises at least 46-80 contiguous nucleotides that are 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 that comprises at least 46-80 contiguous nucleotides that are 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 that comprises at least 46-80 contiguous nucleotides that are 100% identical to any one of SEQ ID NOs: 90, 91, 92, 93, 117, 151, 156-181, and 209-234.

[0241] 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 a variant thereof. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides that are substantially identical to the non-degenerate nucleotides of any one of SEQ ID NOs: 111-114 and 201-204.

[0242] 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: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 75% similarity to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 80% similarity to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 85% similarity to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 90% similarity to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 91% similarity to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 92% similarity to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% similarity to any one of SEQ ID NOs: 111-114 and 201-204.In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 94% identity to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 95% identity to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 96% identity to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 97% identity to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 98% identity to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 99% identity to any one of SEQ ID NOs: 111-114 and 201-204. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity to any one of SEQ ID NOs: 111-114 and 201-204.

[0243] In some embodiments, the guide RNA comprises synthetic or modified nucleotides. In some embodiments, the guide RNA comprises one or more internucleoside linkers modified from natural phosphodiester. In some embodiments, the internucleoside linker of the guide RNA, or all of its contiguous nucleotide sequence, is modified. For example, in some embodiments, the internucleoside linkage comprises sulfur (S), such as a phosphorothioate internucleoside linkage.

[0244] In some embodiments, the guide RNA comprises a modification to the ribose sugar or nucleobase. In some embodiments, the guide RNA comprises one or more nucleosides comprising a modified sugar moiety, where the modified sugar moiety is a modification of the sugar moiety compared to the ribose sugar moiety found in deoxyribose nucleic acids (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, substitution with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g., locked nucleic acids (LNA)), or an unlinked ribose ring, which typically lacks a bond between the C2 and C3 carbons (e.g., UNA). In some embodiments, the sugar-modified nucleoside comprises a bicyclohexose nucleic acid or a tricyclic nucleic acid. In some embodiments, the modified nucleoside comprises a nucleoside in which the sugar moiety is replaced with a non-sugar moiety, such as a peptide nucleic acid (PNA) or morpholino nucleic acid.

[0245] In some embodiments, the guide RNA comprises one or more modified sugars. In some embodiments, sugar modifications include modifications made by changing the substituent on the ribose ring to a group other than hydrogen or to the 2'-OH group naturally found in DNA and RNA nucleosides. In some embodiments, the substituent is introduced at the 2', 3', 4', or 5' position, or a combination thereof. In some embodiments, nucleosides having modified sugar moieties include 2'-modified nucleosides, e.g., 2'-substituted nucleosides. 2'-sugar-modified nucleosides, in some embodiments, are nucleosides having a substituent other than -H or -OH at the 2' position (2'-substituted nucleosides) or include a 2'-linked biradical, and include 2'-substituted nucleosides and LNA (2'-4' biradical 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).

[0246] In some embodiments, the guide RNA comprises one or more modified sugars. In some embodiments, the guide RNA comprises only modified sugars. In certain embodiments, the guide RNA comprises more than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2'-O-methoxyethyl group. In some embodiments, the guide RNA comprises both an internucleoside linker modification and a nucleoside modification.

[0247] In some cases, the guide RNA comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a fungal genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a plant genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a human genomic polynucleotide sequence.

[0248] 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 RNAs are 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 50 The length is about 120, about 50 to about 140, about 50 to about 160, about 50 to about 180, about 50 to about 200, about 50 to about 220, about 50 to about 240, about 100 to about 120, about 100 to about 140, about 100 to about 160, about 100 to about 180, about 100 to about 200, about 100 to about 220, about 100 to about 240, about 160 to about 180, about 160 to about 200, about 160 to about 220, or about 160 to about 240 nucleotides.

[0249] In some cases, the left-hand 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%, at least about 99% identity to any one of any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left-hand 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-hand 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-hand 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-hand 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-hand 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-hand 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-hand 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-hand 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-hand recombinase sequence comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134. In some cases, the left-hand 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-hand 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-hand 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-hand 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-hand 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-hand recombinase sequence comprises a sequence having 100% identity to any one of SEQ ID NOs: 125, 127, 123, 129, 131, 133, 153, and 134.

[0250] 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 comprises a sequence having at least about 95% 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 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 comprises a sequence having 100% identity to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 154, and 155.

[0251] In some cases, the class 2 V-type Cas effector and Tn7-type transposase complex are encoded by a polynucleotide sequence comprising less than about 20 kilobases, less than about 15 kilobases, less than about 10 kilobases, or less than about 5 kilobases.

[0252] MG108 series In some embodiments, provided herein is an MG108 system for transposing a cargo nucleotide sequence into a target nucleic acid site. See FIG. 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 a class 2 V-type Cas effector and an engineered guide polynucleotide configured to hybridize to a target nucleotide sequence. In some embodiments, the class 2 V-type Cas effector comprises a RuvC domain. In some embodiments, the system comprises a Tn7-type transposase complex configured to bind to the Cas effector complex. In some cases, the Tn7-type transposase complex comprises TnsB and TnsC components, but does not comprise TnsA and / or TniQ components.

[0253] In some cases, the cargo nucleotide sequence is adjacent to a left-hand transposase recognition sequence. In some cases, the cargo nucleotide sequence is adjacent to a right-hand transposase recognition sequence. In some cases, the cargo nucleotide sequence is adjacent to a left-hand transposase recognition sequence and a right-hand transposase recognition sequence.

[0254] In some cases, the target nucleic acid comprises a target nucleic acid site. In some cases, the target nucleic acid comprises a PAM sequence that is compatible with a 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.

[0255] In some cases, the engineered guide polynucleotide is configured to bind to a Class 2 type V Cas endonuclease. In some cases, the Class 2 type V Cas effector comprises a polypeptide comprising a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity 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 70% identity to SEQ ID NO: 38 or SEQ ID NO: 108. In some cases, a 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, a 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, a 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, a 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, a 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, a 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 V-type 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, a 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, a 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, a 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, a 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, a 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, a 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 V-type Cas effector comprises a polypeptide comprising a sequence having 100% identity to SEQ ID NO:38 or SEQ ID NO:108.

[0256] In some cases, the Tn7-type transposase complex comprises at least one polypeptide (e.g., at least one, two, three, four, five, six, or more than six 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: 39-40 and 109-110. In some cases, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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 comprises 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 comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises at least one polypeptide comprising a sequence having at least about 94% identity to any one of SEQ ID NOs: 39-40 and 109-110. In some cases, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises at least one polypeptide comprising 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 comprises at least one polypeptide comprising a sequence having 100% identity to any one of SEQ ID NOs: 39-40 and 109-110.

[0257] In some cases, a Tn7-type 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: 39-40 and 109-110. In some cases, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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: 39-40 and 109-110.In some cases, a 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: 39-40 and 109-110. In some cases, a 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: 39-40 and 109-110.

[0258] In some cases, a Tn7-type transposase complex comprises a TnsB component 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: 40 and 109. In some cases, a Tn7-type transposase complex comprises a TnsB component comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 40 and 109. In some cases, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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 comprises 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 comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises a TnsB component comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 40 and 109. In some cases, a Tn7-type transposase complex comprises a TnsB component comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex comprises a TnsB component comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 40 and 109. In some cases, the Tn7-type transposase complex comprises a TnsB component comprising a sequence having 100% identity to any one of SEQ ID NOs: 40 and 109.

[0259] In some cases, a Tn7-type transposase complex comprises a TnsC component 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 any one of SEQ ID NOs: 39 and 110. In some cases, a Tn7-type transposase complex comprises a TnsC component comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 39 and 110. In some cases, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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 comprises 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 comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises a TnsC component comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 39 and 110. In some cases, a Tn7-type transposase complex comprises a TnsC component comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex comprises a TnsC component comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 39 and 110. In some cases, the Tn7-type transposase complex comprises a TnsC component comprising a sequence having 100% identity to any one of SEQ ID NOs: 39 and 110.

[0260] In some cases, the Tn7-type transposase complex comprises TnsB and TnsC components and comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NOs: 40 and 39 or 109 and 110, respectively, or a variant thereof. In some cases, the Tn7-type transposase complex comprises TnsB and TnsC components, each comprising a sequence substantially identical to any one of SEQ ID NOs: 40 and 39 or 109 and 110, or a variant thereof.

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

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

[0263] 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: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 70% of any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 75% of any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 80% of any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 85% of 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 90% similar to any one of SEQ ID NOs: 118, 182, 183, 235, and 236.In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 91% of any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 92% of any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 93% of any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 94% of any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 95% to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 96% to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 97% to any one of SEQ ID NOs: 118, 182, 183, 235, and 236. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 98% 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 99% similar 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 100% identical to any one of SEQ ID NOs: 118, 182, 183, 235, and 236.

[0264] 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 a variant thereof. In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides that are substantially identical to the non-degenerate nucleotides of any one of SEQ ID NOs: 115, 116, 205, and 206.

[0265] 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 has at least about 75% alignment with any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 80% alignment with any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 85% alignment with any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 90% alignment with any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 91% alignment with any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 92% alignment with 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 93% similarity to any one of SEQ ID NOs: 115, 116, 205, and 206.In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 94% alignment with any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 95% alignment with any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 96% alignment with any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 97% alignment with any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 98% alignment with any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 99% alignment with any one of SEQ ID NOs: 115, 116, 205, and 206. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity to any one of SEQ ID NOs: 115, 116, 205, and 206.

[0266] In some embodiments, the guide RNA comprises synthetic or modified nucleotides. In some embodiments, the guide RNA comprises one or more internucleoside linkers modified from natural phosphodiester. In some embodiments, the internucleoside linker of the guide RNA, or all of its contiguous nucleotide sequence, is modified. For example, in some embodiments, the internucleoside linkage comprises sulfur (S), such as a phosphorothioate internucleoside linkage.

[0267] In some embodiments, the guide RNA comprises a modification to the ribose sugar or nucleobase. In some embodiments, the guide RNA comprises one or more nucleosides comprising a modified sugar moiety, where the modified sugar moiety is a modification of the sugar moiety compared to the ribose sugar moiety found in deoxyribose nucleic acids (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, substitution with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g., locked nucleic acids (LNA)), or an unlinked ribose ring, which typically lacks a bond between the C2 and C3 carbons (e.g., UNA). In some embodiments, the sugar-modified nucleoside comprises a bicyclohexose nucleic acid or a tricyclic nucleic acid. In some embodiments, the modified nucleoside comprises a nucleoside in which the sugar moiety is replaced with a non-sugar moiety, such as a peptide nucleic acid (PNA) or morpholino nucleic acid.

[0268] In some embodiments, the guide RNA comprises one or more modified sugars. In some embodiments, sugar modifications include modifications made by changing the substituent on the ribose ring to a group other than hydrogen or to the 2'-OH group naturally found in DNA and RNA nucleosides. In some embodiments, the substituent is introduced at the 2', 3', 4', or 5' position, or a combination thereof. In some embodiments, nucleosides having modified sugar moieties include 2'-modified nucleosides, e.g., 2'-substituted nucleosides. 2'-sugar-modified nucleosides, in some embodiments, are nucleosides having a substituent other than -H or -OH at the 2' position (2'-substituted nucleosides) or include a 2'-linked biradical, and include 2'-substituted nucleosides and LNA (2'-4' biradical 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 comprises one or more modified sugars. In some embodiments, the guide RNA comprises only modified sugars. In certain embodiments, the guide RNA comprises more than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2'-O-methoxyethyl group. In some embodiments, the guide RNA comprises both an internucleoside linker modification and a nucleoside modification.

[0270] In some cases, the guide RNA comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a fungal genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a plant genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a human genomic 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 RNAs are 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 50 The length is about 120, about 50 to about 140, about 50 to about 160, about 50 to about 180, about 50 to about 200, about 50 to about 220, about 50 to about 240, about 100 to about 120, about 100 to about 140, about 100 to about 160, about 100 to about 180, about 100 to about 200, about 100 to about 220, about 100 to about 240, about 160 to about 180, about 160 to about 200, about 160 to about 220, or about 160 to about 240 nucleotides.

[0272] In some cases, the left-hand 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-hand recombinase sequence comprises a sequence having at least about 70% identity to SEQ ID NO: 134. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 75% identity to SEQ ID NO: 134. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 80% identity to SEQ ID NO: 134. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 85% identity to SEQ ID NO: 134. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 90% identity to SEQ ID NO: 134. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 91% identity to SEQ ID NO: 134. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 92% identity to SEQ ID NO: 134. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 93% identity to SEQ ID NO: 134. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 94% identity to SEQ ID NO: 134. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 95% identity to SEQ ID NO: 134. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 96% identity to SEQ ID NO: 134. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 97% identity to SEQ ID NO: 134. In some cases, the left-hand recombinase sequence comprises a sequence having at least about 98% identity to SEQ ID NO: 134.In some cases, the left-hand recombinase sequence comprises a sequence having at least about 99% identity to SEQ ID NO: 134. In some cases, the left-hand recombinase sequence comprises a sequence having 100% identity to SEQ ID NO: 134.

[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: 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 comprises a sequence having at least about 80% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence comprises a sequence having at least about 85% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence comprises a sequence having at least about 90% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence comprises a sequence having at least about 91% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence comprises a sequence having at least about 92% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence comprises a sequence having at least about 93% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence comprises a sequence having at least about 94% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence comprises a sequence having at least about 95% identity to SEQ ID NO: 135. In some cases, the right recombinase sequence comprises 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.

[0274] In some cases, the class 2 V-type Cas effector and Tn7-type transposase complex are encoded by a polynucleotide sequence comprising less than about 20 kilobases, less than about 15 kilobases, less than about 10 kilobases, or less than about 5 kilobases.

[0275] MG110 series In some embodiments, provided herein is an MG110 system for transposing a cargo nucleotide sequence into a target nucleic acid site. 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 a class I type I Cas effector and an engineered guide polynucleotide configured to hybridize to a target nucleotide sequence. In some embodiments, the system comprises a Tn7-type transposase complex configured to bind to the Cas effector complex.

[0276] In some cases, the cargo nucleotide sequence is adjacent to a left-hand transposase recognition sequence. In some cases, the cargo nucleotide sequence is adjacent to a right-hand transposase recognition sequence. In some cases, the cargo nucleotide sequence is adjacent to a left-hand transposase recognition sequence and a right-hand transposase recognition sequence.

[0277] In some cases, the target nucleic acid comprises a target nucleic acid site. In some cases, the target nucleic acid comprises a PAM sequence that is compatible with a 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. In some cases, the PAM sequence is located 5' of the target nucleic acid site.

[0278] In some cases, the engineered guide polynucleotide is configured to bind to a Class 1 Type I Cas endonuclease. In some cases, the Class 1 Type I Cas effector comprises a polypeptide comprising a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs:41-43 and 48-50. In some cases, a Class 1 Type I Cas effector comprises a polypeptide having a sequence at least about 70% identical to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, a Class 1 Type I Cas effector comprises a polypeptide having a sequence at least about 75% identical to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, a Class 1 Type I Cas effector comprises a polypeptide having a sequence at least about 80% identical to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, a Class 1 Type I Cas effector comprises a polypeptide having a sequence at least about 85% identical to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, a Class 1 Type I Cas effector comprises a polypeptide having a sequence at least about 90% 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 having a sequence at least about 91% 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 having a sequence at least about 92% identical to any one of SEQ ID NOs: 41-43 and 48-50.In some cases, a Class 1 Type I Cas effector comprises a polypeptide having a sequence at least about 93% identical to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, a Class 1 Type I Cas effector comprises a polypeptide having a sequence at least about 94% identical to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, a Class 1 Type I Cas effector comprises a polypeptide having a sequence at least about 95% identical to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, a Class 1 Type I Cas effector comprises a polypeptide having a sequence at least about 96% identical to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, a Class 1 Type I Cas effector comprises a polypeptide having a sequence at least about 97% identical to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, a Class 1 Type I Cas effector comprises a polypeptide having a sequence at least about 98% identical to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, a Class 1 Type I Cas effector comprises a polypeptide having a sequence at least about 99% identical to any one of SEQ ID NOs: 41-43 and 48-50. In some cases, a Class 1 Type I Cas effector comprises a polypeptide having a sequence 100% identical to any one of SEQ ID NOs: 41-43 and 48-50.

[0279] In some cases, the engineered guide polynucleotide is configured to bind to a Class 1 Type I Cas endonuclease. In some cases, Class 1 Type I Cas effectors include 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.

[0280] In some cases, the Tn7-type transposase complex comprises at least one polypeptide (e.g., at least one, two, three, four, five, six, or more than six 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:44-47 and 51-54. In some cases, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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 comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises 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, a Tn7-type transposase complex comprises at least one polypeptide comprising a sequence having 100% identity to any one of SEQ ID NOs: 44-47 and 51-54.

[0281] In some cases, a Tn7-type 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: 44-47 and 51-54. In some cases, a 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, a 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, a 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, a 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 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: 44-47 and 51-54.In some cases, a 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: 44-47 and 51-54. In some cases, a 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: 44-47 and 51-54. In some cases, a 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: 44-47 and 51-54. In some cases, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a Tn7-type transposase complex comprises TnsA, TnsB, TnsC, and TniQ components.

[0282] In some embodiments, the systems disclosed herein comprise at least one engineered guide polynucleotide, e.g., a gRNA.

[0283] In some embodiments, provided herein are engineered guide polynucleotides, such as guide RNAs (gRNAs).

[0284] In some cases, the engineered guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides that has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 70% of the sequence of any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 75% of the sequence of any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 80% of the sequence of any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 85% of the sequence of any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 90% of any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 91% of any one of SEQ ID NOs: 121, 122, 207, and 208.In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 92% of the sequence of any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 93% of the sequence of any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 94% of the sequence of any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are at least about 95% of the sequence of any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that comprises at least 46-80 contiguous nucleotides that are 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 that comprises at least 46-80 contiguous nucleotides that are 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 that comprises at least 46-80 contiguous nucleotides that are 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 that comprises at least 46-80 contiguous nucleotides that are 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.

[0285] 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 has at least about 75% alignment with any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 80% alignment with any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 85% alignment with any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 90% alignment with any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 91% alignment with any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 92% alignment with 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 93% similarity to any one of SEQ ID NOs: 121, 122, 207, and 208.In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 94% alignment with any one of SEQ ID NOs: 121, 122, 207, and 208. In some embodiments, the engineered guide polynucleotide comprises a sequence that has at least about 95% alignment with any one of SEQ ID...

Claims

1. 1. A system for translocating a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid, comprising: a) a Cas effector complex comprising a class 2 type II 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 recombinase or transposase complex configured to bind to said Cas effector complex; and c) a double-stranded nucleic acid configured to interact with said recombinase or transposase complex and comprising said cargo nucleotide sequence; d) a functional domain comprising a DNA binding domain (DBD) or a chromatin modulation domain (CMD).

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

3. The system described in claim 1, wherein the Cas effector complex is fused to the recombinase or transposase complex.

4. A system described in any one of claims 1 to 3, wherein the cargo nucleotide sequence is adjacent to a left transposase recognition sequence and a right transposase recognition sequence recognized by the recombinase or transposase complex.

5. The system described in claim 4, wherein the left recombinase sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 17-18, and the right recombinase sequence comprises a sequence having at least 80% identity to SEQ ID NO:

19.

6. The system described in claim 1, further comprising a PAM sequence compatible with the Cas effector complex, the PAM sequence being located approximately 50 to approximately 70 base pairs from the target nucleic acid site.

7. The system described in claim 6, wherein the PAM sequence is located 3' of the target nucleic acid site or 5' of the target nucleic acid site.

8. The system described in claim 1, wherein the class 2 type II Cas effector comprises a polypeptide having an array having at least 80% identity to sequence number 1.

9. The system described in claim 1, wherein the recombinase or transposase complex is a Tn7-type transposase complex.

10. The system described in claim 1, 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 to 5.

11. The system described in claim 1, wherein the engineered guide polynucleotide comprises a sequence comprising at least about 46 to 80 consecutive nucleotides having at least 80% identity to SEQ ID NO:

12.

12. The system described in claim 1, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity with sequence number 11.

13. The system described in claim 1, wherein the functional domain is derived from human histone 1 central globular domain, HMGN1, cbx5, or Saccharolobus solfataricus sso7d.

14. The system described in claim 1, wherein the functional domain comprises a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 264 to 266.

15. The system described in claim 1, wherein the class 2 type II Cas effector is fused to the functional domain to form a fusion protein.

16. The system described in claim 1, wherein the recombinase or transposase complex comprises a TniQ protein having at least 80% sequence identity with the TniQ domain of SEQ ID NO:

270.

17. The system of claim 16, wherein the TniQ protein is fused to the functional domain to form a fusion protein.

18. The system described in claim 1, 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 or 620.

19. The system described in claim 1, wherein the class 2 type II Cas effector and the recombinase or transposase complex are encoded by a polynucleotide sequence comprising less than about 10 kilobases.

20. A method for translocating a cargo nucleotide sequence within a target nucleic acid site in a target nucleic acid, comprising introducing the system described in claim 1 into a cell.

21. A cell comprising the system described in claim 1.

22. The cell of claim 21, wherein the cell is a eukaryotic cell, a mammalian cell, an immortalized cell, an insect cell, a yeast cell, a plant cell, a fungal cell, or a prokaryotic cell.

23. The cell of claim 21, 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, Saos, C2C12, L cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof.

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