CRISPR-associated transposases and uses thereof

Patent metadata

Field	Value
Publication number	US-10995327-B2
Application number	US-201616066996-A
Country	US
Kind code	B2
Filing date	Dec 29, 2016
Priority date	Dec 29, 2015
Publication date	May 4, 2021
Grant date	May 4, 2021

Abstract

Provided herein are systems, methods, and compositions for the modification of target DNA sequences. More particularly, systems, methods, and compositions for editing genomic DNA in eukaryotic cells with a CRISPR-associated transposase are provided. Also provided are vectors and vector systems which encode one or more CRISPR-associated transposases, as well as methods for the design and use of such vectors. Also provided are methods for identifying and validating novel CRISPR-associated transposases.

First claim

What is claimed is:

1. A recombinant nucleic acid, comprising a heterologous promoter operably linked to a polynucleotide encoding a CRISPR-associated transposase with an amino acid sequence selected from the group consisting of SEQ ID NOs: 124-133, 135-246 and 275-287.

2. The recombinant nucleic acid of claim 1 , wherein the CRISPR-associated transposase:
a. is from a bacterium selected from the group consisting of Lysinibacillus sp., Brevibacillus sp., Sphingobium sp., Undibacterium sp., Bacillus sp., Chryseobacterium sp., Sphingomonas sp., Labrys sp., Paenibacillus sp., Streptomyces sp., and Stenotrophomonas sp.;
b. is from a bacterium selected from the group consisting of Brevibacillus brevis, Brevibacillus laterosporus, Brevibacillus parabrevis, Bacillus megaterium, Bacillus thuringiensis, Bacillus weihenstephanensis, Enterococcus faecalis, Undibacterium pigrum, Novosphingobium rosa, Labrys methylaminiphilus, Paenibacillus thiaminolyticus, Paenibacillus lentimorbus , and Paenibacillus terrae;
c. is associated in the bacterial genome with a CRISPR locus;
d. is located in the same operon as a CRISPR locus;
e. is located within 2.5 kilobases of a CRISPR locus;
f. is encoded by a nucleotide sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-123, 604-627 and 2020-3379; or
g. any combination of (a), (b), (c), (d), (e), and (f).

3. The recombinant nucleic acid of claim 1 , further comprising at least one polynucleotide encoding a guide RNA capable of hybridizing with a target sequence, wherein the guide RNA forms a complex with the CRISPR-associated transposase.

4. The recombinant nucleic acid of claim 1 , further comprising at least one polynucleotide encoding a donor polynucleotide.

5. The recombinant nucleic acid of claim 1 , wherein the polynucleotide encoding the CRISPR-associated transposase further encodes at least one nuclear localization signal (NLS).

6. A vector comprising the recombinant nucleic acid of claim 1 .

7. A eukaryotic cell comprising the recombinant nucleic acid of claim 1 .

8. A non-naturally occurring system for sequence-specific modification of a target nucleic acid sequence, comprising
(a) one or more guide RNAs or a DNA molecule encoding the one or more guide RNAs, wherein the one or more guide RNAs are capable of hybridizing with the target nucleic acid sequence, and
(b) a CRISPR-associated transposase having an amino acid sequence selected from the group consisting of SEQ ID NO: 124-133, 135-246 and 275-287 or polynucleotide encoding the CRISPR-associated transposase,
wherein the one or more guide RNAs and the CRISPR-associated transposase do not naturally occur together.

9. The system of claim 8 ,
(a) wherein the target nucleic acid sequence comprises a coding nucleic acid sequence, a non-coding nucleic acid sequence, or a combination of a coding and a non-coding nucleic acid sequence; or
(b) wherein the target nucleic acid sequence comprises an endogenous gene or a transgene; or
(c) any combination of (a) and (b).

10. The system of claim 8 , wherein the system comprises a divalent cation.

11. The system of claim 8 , further comprising a donor polynucleotide.

12. A method for sequence-specific modification of a target nucleic acid sequence in a cell, comprising providing the system of claim 8 to a cell that comprises the target nucleic acid sequence.

13. A method for sequence-specific modification of a target nucleic acid sequence in a cell, comprising providing to the cell a CRISPR-associated transposase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 124-133, 135-246 and 275-287, whereby the target nucleic acid sequence is modified.

14. The method of claim 13 , further comprising a guide RNA capable of associating with the CRISPR-associated transposase and hybridizing with the target nucleic acid sequence.

15. The method of claim 14 , wherein:
(a) the guide RNA is provided by expressing in the cell a recombinant DNA molecule encoding the guide RNA;
(b) the CRISPR-associated transposase is provided by expressing in the cell a recombinant DNA molecule encoding the CRISPR-associated transposase; or
(c) both (a) and (b).

16. The method of claim 14 , wherein:
(a) the guide RNA is provided by contacting the cell with a composition comprising the guide RNA or a recombinant DNA molecule encoding the guide RNA;
(b) the CRISPR-associated transposase is provided by contacting the cell with a composition comprising the CRISPR-associated transposase or a recombinant DNA molecule encoding the CRISPR-associated transposase; or
(c) the CRISPR-associated transposase is complexed with the guide RNA and is provided to the cell as a particle.

17. The method of claim 13 , further comprising providing a donor polynucleotide to the cell.

18. A eukaryotic cell containing the non-naturally occurring system of claim 8 .

Assignees

• Monsanto Technology Llc

Inventors

• Chittoor Jaishree M
• Nagy Ervin

Classifications

• C12N15/8241

  Phenotypically and genetically modified plants via recombinant DNA technology · CPC title

• C12N15/102 Primary

  Mutagenizing nucleic acids · CPC title

• C12N9/22 Primary

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

• C12N2800/80

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