Novel crispr-associated transposon systems and components

Patent metadata

Field	Value
Publication number	US-2020291395-A1
Application number	US-201816761206-A
Country	US
Kind code	A1
Filing date	Nov 2, 2018
Priority date	Nov 2, 2017
Publication date	Sep 17, 2020
Grant date	—

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, enginceled 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

1 . An engineered, non-naturally occurring Clustered Interspaced Short Palindromic Repeat (CRISPR)—Cas system of CLUST.004377 comprising:
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, and any one of the following:
a. a CRISPR-associated protein containing charged N-terminal residues that are capable of binding to the Guide, either as a monomer or multimer, and of targeting the target nucleic acid sequence complementary to the Guide spacer;
b. a CRISPR-associated protein containing charged N-terminal residues and an HTH domain-containing protein that together are capable of binding to the Guide and of targeting the target nucleic acid sequence complementary to the spacer sequence;
c. a CRISPR-associated protein containing charged N-terminal residues and a transposase module that together are capable of binding to the Guide and of targeting the target nucleic acid sequence complementary to the spacer sequence;
d. a CRISPR-associated protein containing charged N-terminal residues, an HTH domain-containing protein, and a transposase module that together are capable of binding to the Guide and of targeting the target nucleic acid sequence complementary to the spacer sequence; and
e. a payload nucleic acid flanked by transposon end sequences.

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 . The system of claim 1 , wherein targeting of the target nucleic acid by the protein and the Guide results in a modification in the target nucleic acid, which optionally is a double-stranded cleavage event or a single-stranded cleavage event.

5 - 6 . (canceled)

7 . The system of claim 1 , further comprising a donor template nucleic acid, which optionally is a DNA.

8 . (canceled)

9 . The system of claim 1 , wherein the target nucleic acid is a double-stranded DNA and the targeting of the double-stranded DNA results in scarless DNA insertion.

10 . The system of claim 1 , wherein the modification results in cell toxicity.

11 . The system of claim 1 , within a cell, which is a eukaryotic cell or a prokaryotic cell.

12 - 13 . (canceled)

14 . 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.

15 . 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.

16 . 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.

17 . The system of claim 1 , wherein the transposase module comprises Mu-transposase, TniQ, and/or TniB.

18 . The system of claim 1 , wherein the CRISPR-associated protein comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to an amino acid sequence provided in any one of Tables 2-6.

19 . The system of claim 1 , wherein the crRNA or Guide comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to a nucleic acid sequence provided in Table 7.

20 . The system of claim 1 , wherein the payload nucleic acid is flanked by transposon end sequences comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to a nucleic acid sequence provided in Table 8 or Table 9.

21 . (canceled)

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

23 . (canceled)

24 . 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.

25 - 28 . (canceled)

29 . The system of claim 4 , 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 heroes simplex vector.

30 - 31 . (canceled)

32 . 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.

33 . (canceled)

34 . 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.

35 - 41 . (canceled)

Assignees

• Arbor Biotechnologies Inc

Inventors

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

Classifications

• 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

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N9/1241

  Nucleotidyltransferases (2.7.7) · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title