CRISPR DNA targeting enzymes and systems

Patent metadata

Field	Value
Publication number	US-11912992-B2
Application number	US-202218064171-A
Country	US
Kind code	B2
Filing date	Dec 9, 2022
Priority date	Mar 14, 2018
Publication date	Feb 27, 2024
Grant date	Feb 27, 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 nucleic acids such as DNA. 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: 5, and a nuclear localization sequence (NLS) having an amino acid sequence having at least 90% identical to KRPAATKKAGQAKKKK (SEQ ID NO: 301);
wherein the CRISPR-Cas effector protein binds to the RNA guide, and wherein the spacer sequence is complementary to at least 15 nucleotides of a target nucleic acid.

2. The system of claim 1 , wherein the NLS has an amino acid sequence according to KRPAATKKAGQAKKKK (SEQ ID NO: 301).

3. The system of claim 1 , wherein the CRISPR-Cas effector protein comprises one or more of:
(a) a RuvC domain comprising the amino acid sequence X 1 SHX 4 DX 6 X 7 (SEQ ID NO: 200, wherein X 1 is S or T, X 4 is Q or L, X 6 is P or S, and X 7 is F or L,
(b) a RuvC domain comprising the amino acid sequence X 1 XDXNX 6 X 7 XXXX 11 (SEQ ID NO: 201), wherein X 1 is A, G, or S, X is any amino acid, X 6 is Q or I, X 7 is T, S, or V, and X 11 is T or A; and
(c) a RuvC domain comprising the amino acid sequence X 1 X 2 X 3 E (SEQ ID NO: 210), wherein X 1 is C, F, I, L, M, P, V, W, or Y, X 2 is C, F, I, L, M, P, R, V, W, or Y, and X 3 is C, F, G, I, L, M, P, V, W, or Y.

4. The system of claim 1 , wherein the CRISPR-Cas effector protein comprises the amino acid sequence set forth in SEQ ID NO: 5.

5. The system of claim 1 , wherein the direct repeat sequence comprises any one of:
(a) 5′-CCGUCNNNNNNUGACGG-3′ (SEQ ID NO: 202) proximal to the 3′ end, wherein N is any nucleobase; or
(b) 5′-UCX 3 UX 5 X 6 X 7 UUGACGG-3′ (SEQ ID NO: 205) proximal to the 3′ end, wherein X 3 is C, T, or U, X 5 is A, T, or U, X 6 is A, C, or G, and X 7 is A or G.

6. The system of claim 1 , wherein the RNA guide comprises a nucleotide sequence with at least 95% sequence identity to SEQ ID NO: 101.

7. The system of claim 1 , wherein the RNA guide comprises a nucleotide sequence set forth in SEQ ID NO: 101.

8. The system of claim 1 , wherein the direct repeat sequence comprises an RNA transcript of a nucleotide sequence with at least 95% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10.

9. The system of claim 1 , wherein the direct repeat sequence comprises an RNA transcript of a nucleotide sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10.

10. The system of claim 1 , wherein the direct repeat sequence comprises a stem-loop structure proximal to a 3′ end of the direct repeat sequence, wherein the stem-loop structure comprises:
(a) a first stem nucleotide strand 5 nucleotides in length;
(b) a second stem nucleotide strand 5 nucleotides in length, wherein the first and second stem nucleotide strands bind with each other; and
(c) a loop nucleotide strand arranged between the first and second stem nucleotide strands, wherein the loop nucleotide strand comprises 6, 7, or 8 nucleotides.

11. The system of claim 1 , wherein the spacer sequence comprises between 15 and 47 nucleotides in length.

12. The system of claim 11 , wherein the spacer sequence comprises between 24 and 38 nucleotides in length or between 20 and 33 nucleotides in length.

13. The system of claim 11 , wherein the spacer sequence has at least 90%, 95%, or 100% complementarity to the target nucleic acid.

14. The system of claim 1 , wherein the NLS is attached to the C-terminus of the CRISPR-Cas effector protein.

15. The system of claim 1 , wherein the NLS is attached to the N-terminus of the CRISPR-Cas effector protein.

16. 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′-TTN-3′, wherein N is any nucleotide.

17. The system of claim 16 , wherein the PAM sequence comprises a nucleotide sequence set forth as 5′-TTY-3′, wherein Y is C or T, or 5′-TTH-3′, wherein H is A or C or T.

18. The system of claim 1 , wherein the CRISPR-Cas effector protein further comprises a second NLS.

19. 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.

20. The system of claim 1 , wherein the CRISPR-Cas effector protein further includes a linker sequence.

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

22. The system of claim 21 , wherein the vector comprises a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector.

23. 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.

24. A cell comprising the system of claim 1 .

25. The cell of claim 24 , wherein the cell is a human cell.

26. A method of binding the system of claim 1 to the target nucleic acid in a cell comprising:
(a) providing the system; and
(b) delivering the system to the 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.

27. The method of claim 26 , wherein the target nucleic acid is a double-stranded DNA.

28. The method of claim 26 , wherein binding the system to the target nucleic acid results in cleavage, or a formation of an insertion or a deletion the target nucleic acid.

Assignees

• Arbor Biotechnologies Inc

Inventors

• Chong Shaorong
• Yan Winston X
• Scott David A
• Cheng David R
• Hunnewell Pratyusha

Classifications

• C12N15/11 Primary

  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

• C12N9/22 Primary

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

• C12N15/902

  using homologous recombination · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N2310/531

  Stem-loop; Hairpin · CPC title