Systems and methods for rearranging cargo nucleotide sequences

Abstract

The present disclosure provides systems and methods for translocating a cargo nucleotide sequence to a target nucleic acid site. These systems and methods may include a first double-stranded nucleic acid comprising a cargo nucleotide sequence configured to interact with a recombinase complex, a cas effector complex comprising a cas effector and at least one engineered guide polynucleotide configured to hybridize to the target nucleic acid site, and a recombinase complex configured to recruit the cargo nucleotide to the target nucleic acid site.

Claims

1. A manipulated nuclease system, An endonuclease containing a RuvC domain, which is a class II, type V-K Cas effector having at least 90% identity with SEQ ID NO: 1, A manipulated guide ribonucleic acid (RNA) comprising a spacer sequence configured to form a complex with the endonuclease and to hybridize to a target nucleic acid sequence, and A modified nuclease system, including [specific component].

2. The manipulated nuclease system according to claim 1, wherein the endonuclease comprises a sequence having at least 90% sequence identity with SEQ ID NO:

1.

3. The manipulated nuclease system according to claim 1 or 2, wherein the endonuclease comprises SEQ ID NO:

1.

4. The manipulated nuclease system according to any one of claims 1 to 3, wherein the manipulated guide RNA comprises a sequence having at least 90% identity with any one of sequence numbers 6, 32-33, 94-95, or 104-105, comprising at least about 46 to 80 consecutive nucleotides.

5. The manipulated nuclease system according to any one of claims 1 to 3, wherein the manipulated guide RNA comprises a sequence having at least 46 to 80 consecutive nucleotides having at least 90% identity with either SEQ ID NO: 5 or 6.

6. The manipulated nuclease system according to any one of claims 1 to 3, wherein the manipulated guide RNA comprises a sequence having at least 90% sequence identity with respect to any one of the undegenerate nucleotides of sequence numbers 106 to 108.

7. The manipulated nuclease system according to any one of claims 1 to 6, wherein the manipulated guide RNA comprises a sequence having at least 90% sequence identity with respect to one of the undegenerate nucleotides of sequence number 5, 45-63, 68-75, or 96-103.

8. The manipulated nuclease system according to any one of claims 1 to 7, wherein the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence containing sequence number 31.

9. A double-stranded nucleic acid containing a cargo nucleotide sequence configured to interact with a Tn7 type transposase complex, A Cas effector complex comprising a class II, type V Cas effector and an engineered guide polynucleotide encoding an engineered guide RNA configured to hybridize to a target nucleic acid site, The Tn7 type transposase complex configured to bind to the Cas effector complex, comprising a Tn7 type transposase complex and a Tn7 type transposase complex comprising a TnsB subunit. A system for rearranging the cargo nucleotide sequence to the target nucleic acid site, comprising: A system comprising a class II, type V Cas effector containing a polypeptide having at least 90% identity with sequence number 1.

10. The system according to claim 9, wherein the Class II, Type V Cas effector comprises a polypeptide having at least 90% identity with SEQ ID NO:

1.

11. The system according to claim 9 or 10, wherein the Class II, Type V Cas effector comprises a polypeptide containing Sequence ID No.

1.

12. The system according to any one of claims 9 to 11, wherein the TnsB subunit comprises a polypeptide having a sequence having at least 90% sequence identity with any one of sequence numbers 2, 13, 17, or 65.

13. The system according to any one of claims 9 to 12, wherein the TnsB subunit comprises a polypeptide having a sequence having at least 90% sequence identity with respect to sequence number 2.

14. The system according to any one of claims 9 to 13, wherein the Tn7 type transposase complex comprises a polypeptide having a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 3-4, 14-15, 18-19, or 66-67.

15. The system according to any one of claims 9 to 14, wherein the Tn7 type transposase complex comprises a polypeptide having at least 90% sequence identity with SEQ ID NO: 3 or 4.

16. The system according to any one of claims 9 to 15, wherein the manipulated guide polynucleotide comprises a sequence having at least 90% sequence identity with respect to one of the non-degenerate nucleotides, SEQ ID NOs. 5, 45-63, 68-75, 96-103, 106, 107, or 108.

17. The system according to any one of claims 9 to 16, wherein the manipulated guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs. 5-6, 32-33, 94-95, or 104-105, comprising at least about 46-80 consecutive nucleotides.

18. The system according to any one of claims 9 to 17, wherein the manipulated guide polynucleotide comprises a sequence having at least 90% sequence identity with respect to either SEQ ID NO: 5 or 6, and comprising at least about 46 to 80 consecutive nucleotides.

19. The system according to any one of claims 9 to 18, wherein the cargo nucleotide sequence is adjacent to the left transposase recognition sequence and the right transposase recognition sequence.

20. The system according to claim 19, wherein the transposase recognition sequence on the left side includes a sequence having at least 90% identity with any one of sequence numbers 9, 11, 36-38, 76, or 78.

21. The system according to claim 19 or 20, wherein the transposase recognition sequence on the right side includes a sequence having at least 90% identity with any one of sequence numbers 8, 10, 39-44, 77, 79, or 93.

22. A method for modifying a target nucleotide sequence, comprising the step of contacting the target nucleotide sequence in vitro with the system described in any one of claims 9 to 21.

23. The method according to claim 22, wherein the target nucleotide sequence is located within a cell.

24. One or more nucleic acids encoding the system described in any one of claims 9 to 21.

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