CRISPR DNA targeting enzymes and systems

Patent metadata

Field	Value
Publication number	US-11795442-B2
Application number	US-202217820444-A
Country	US
Kind code	B2
Filing date	Aug 17, 2022
Priority date	Sep 9, 2019
Publication date	Oct 24, 2023
Grant date	Oct 24, 2023

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 nucleic acids. Each system includes one or more protein components and one or more nucleic acid components that together target nucleic acids.

First claim

What is claimed is:

1. An engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated (Cas) system comprising:
(a) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence; and
(b) a CRISPR-Cas effector protein or a nucleic acid encoding the CRISPR-Cas effector protein, wherein the CRISPR-Cas effector protein comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 4,
wherein the CRISPR-Cas effector protein binds to the RNA guide, and wherein the spacer sequence is capable of binding to a target nucleic acid in a eukaryotic cell.

2. The system of claim 1 , wherein the CRISPR-Cas effector protein comprises an amino acid sequence with at least 99% identity to SEQ ID NO: 4.

3. The system of claim 1 , wherein the direct repeat sequence comprises a nucleotide sequence with at least 95% sequence identity to SEQ ID NO: 291.

4. The system of claim 3 , wherein the direct repeat sequence comprises the nucleotide sequence of SEQ ID NO: 291.

5. The system of claim 1 , wherein the spacer sequence comprises between 15 and 55 nucleotides in length.

6. The system of claim 5 , wherein the spacer sequence comprises between 20 and 45 nucleotides in length.

7. The system of claim 1 , wherein the target nucleic acid comprises a sequence complementary to a nucleotide sequence in the spacer sequence.

8. The system of claim 1 , wherein the system does not include a tracrRNA.

9. The system of claim 1 , wherein the CRISPR-Cas effector protein recognizes a protospacer adjacent motif (PAM) sequence, wherein the PAM sequence comprises a nucleotide sequence set forth as 5′-NTTN-3′, 5′-NTTR-3′, or 5′-NNR-3′, wherein “N” is any nucleotide and “R” is A or G.

10. The system of claim 1 , wherein the CRISPR-Cas effector protein further comprises at least one nuclear localization signal (NLS), at least one nuclear export signal (NES), or at least one NLS and at least one NES.

11. The system of claim 1 , wherein the CRISPR-Cas effector protein further comprises a peptide tag, a fluorescent protein, a base-editing domain, a DNA methylation domain, a histone residue modification domain, a localization factor, a transcription modification factor, a light-gated control factor, a chemically inducible factor, or a chromatin visualization factor.

12. The system of claim 1 , wherein the nucleic acid encoding the CRISPR-Cas effector protein is codon-optimized for expression in a eukaryotic cell.

13. The system of claim 1 , wherein the nucleic acid encoding the CRISPR-Cas effector protein is operably linked to a promoter.

14. The system of claim 1 , wherein the nucleic acid encoding the CRISPR-Cas effector protein is in a vector.

15. The system of claim 14 , wherein the vector is a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector.

16. The system of claim 1 , wherein the system is present in a delivery system comprising a nanoparticle, a liposome, an exosome, a microvesicle, or a gene-gun.

17. An isolated eukaryotic cell comprising the system of claim 1 .

18. The cell of claim 17 , wherein the cell is a mammalian cell or a plant cell.

19. The cell of claim 18 , wherein the cell is an isolated human cell.

20. A method of binding the system of claim 1 to the target nucleic acid of claim 1 comprising:
(a) providing the system of claim 1 ; and
(b) delivering the system to a eukaryotic cell, wherein the cell comprises the target nucleic acid, wherein the CRISPR-Cas effector protein binds to the RNA guide, and wherein the spacer sequence binds to the target nucleic acid.

21. The method of claim 20 , wherein the target nucleic acid is a single-stranded DNA or a double-stranded DNA.

22. The method of claim 20 , wherein binding the system to the target nucleic acid results in cleavage of the target nucleic acid.

23. The method of claim 22 , wherein cleavage of the target nucleic acid results in formation of an insertion or a deletion in the target nucleic acid.

Assignees

• Arbor Biotechnologies Inc

Inventors

• Scott David A
• Cheng David R
• Yan Winston X
• Ditommaso Tia M

Classifications

• C12N9/22 Primary

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

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12N5/0687

  Renal stem cells; Renal progenitors · 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

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title