CRISPR-associated transposon systems and components

Patent metadata

Field	Value
Publication number	US-12054754-B2
Application number	US-201816761206-A
Country	US
Kind code	B2
Filing date	Nov 2, 2018
Priority date	Nov 2, 2017
Publication date	Aug 6, 2024
Grant date	Aug 6, 2024

Abstract

The disclosure describes novel systems, methods, and compositions for the manipulation of nucleic acids in a targeted fashion. The disclosure describes non-naturally occurring, engineered CRISPR systems, components, and methods for targeted modification of DNA, RNA, and protein substrates. Each system includes one or more protein components and one or more nucleic acid components that together target DNA, RNA, or protein substrates.

First claim

What is claimed is:

1. An engineered, non-naturally occurring Clustered Interspaced Short Palindromic Repeat (CRISPR)-Cas system comprising:
(a) a Guide consisting of a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid, wherein the Guide is CRISPR RNA (crRNA) or DNA;
(b) a CRISPR-associated protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 19;
(c) a CRISPR-associated protein comprising an rve integrase domain;
(d) a CRISPR-associated protein comprising a TniQ domain; and
(e) a CRISPR-associated protein comprising a TniB domain.

2. The system of claim 1 , wherein the target nucleic acid is a DNA or an RNA.

3. The system of claim 2 , wherein the target nucleic acid is double-stranded DNA.

4. A method of targeting and editing a target nucleic acid, the method comprising contacting the target nucleic acid with a system of claim 1 , wherein optionally the method results in an insertion or substitution of DNA to correct a native locus.

5. A method of targeting the insertion of a payload nucleic acid at a site of a target nucleic acid, the method comprising contacting the target nucleic acid with a system of claim 1 , wherein optionally the method results in a targeted insertion or deletion of a DNA payload into a specific genomic target site.

6. A method of targeting the excision of a payload nucleic acid from a site at a target nucleic acid, the method comprising contacting the target nucleic acid with a system of claim 1 , wherein optionally the method results in a targeted deletion of DNA to correct a native locus.

7. The system of claim 1 , wherein the system comprises a Mu-transposase.

8. The system of claim 1 , wherein the CRISPR-associated protein comprises at least one nuclear localization signal or at least one nuclear export signal.

9. The system of claim 1 , wherein at least one component of the system is encoded by a codon-optimized nucleic acid for expression in a cell, which optionally is present within at least one vector, which optionally comprises one or more regulatory elements operably-linked to a nucleic acid encoding the component of the system, wherein the one or more regulatory elements optionally comprises at least one promoter, which optionally comprises an inducible promoter or a constitutive promoter.

10. The system of claim 9 , wherein the at least one vector comprises a plurality of vectors, and/or is a viral vector that is optionally selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated vector, and a herpes simplex vector.

11. The system of claim 1 , wherein the system is present in a delivery system, which optionally comprises a delivery vehicle selected from the group consisting of a liposome, an exosome, a microvesicle, and a gene-gun.

12. A cell comprising the system of claim 1 , wherein optionally the cell is a eukaryotic cell, which optionally is a mammalian cell, such as a human cell, or a plant cell; or is a prokaryotic cell.

13. The system of claim 1 , wherein:
(a) the CRISPR-associated protein comprising an rve integrase domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 54;
(b) the CRISPR-associated protein comprising a TniQ domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 80; and
(c) the CRISPR-associated protein comprising a TniB domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 103.

14. The system of claim 1 , further comprising a payload nucleic acid flanked by transposon end sequences.

15. The system of claim 14 , wherein at least one of the transposon end sequences comprises SEQ ID NO: 142.

16. The system of claim 14 , wherein at least one of the transposon end sequences comprises SEQ ID NO: 141.

17. The system of claim 14 , wherein at least one of the transposon end sequences comprises at least 100 contiguous nucleotides of SEQ ID NO: 177.

18. The system of claim 1 , wherein:
(a) the CRISPR-associated protein comprising an rve integrase domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 54;
(b) the CRISPR-associated protein comprising a TniQ domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 80; and
(c) the CRISPR-associated protein comprising a TniB domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 103.

19. The system of claim 1 , wherein:
(a) the CRISPR-associated protein comprising an rve integrase domain comprises the amino acid sequence of SEQ ID NO: 54;
(b) the CRISPR-associated protein comprising a TniQ domain comprises the amino acid sequence of SEQ ID NO: 80; and
(c) the CRISPR-associated protein comprising a TniB domain comprises the amino acid sequence of SEQ ID NO: 103.

20. The system of claim 1 , comprising a CRISPR-associated protein comprising an amino acid sequence that is 100% identical to SEQ ID NO: 19.

21. The system of claim 1 , comprising:
(a) the CRISPR-associated protein comprising an rve integrase domain comprises an amino acid sequence that is 100% identical to SEQ ID NO: 19;
(b) the CRISPR-associated protein comprising an amino acid sequence that is 100% identical to SEQ ID NO: 54;
(c) the CRISPR-associated protein comprising a TniQ domain comprises an amino acid sequence that is 100% identical to SEQ ID NO: 80; and
(d) the CRISPR-associated protein comprising a TniB domain comprises an amino acid sequence that is 100% identical to SEQ ID NO: 103.

Assignees

• Arbor Biotechnologies Inc

Inventors

• Scott David A
• Cheng David R
• Yan Winston X

Classifications

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N15/1058

  Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms · CPC title

• C12N9/1241

  Nucleotidyltransferases (2.7.7) · CPC title

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title