CRISPR DNA targeting enzymes and systems

Patent metadata

Field	Value
Publication number	US-10808245-B2
Application number	US-201916680104-A
Country	US
Kind code	B2
Filing date	Nov 11, 2019
Priority date	Mar 14, 2018
Publication date	Oct 20, 2020
Grant date	Oct 20, 2020

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 Cluster 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 the amino acid sequence set forth in SEQ ID NO: 5,
wherein the CRISPR-Cas effector protein binds to the RNA guide, and wherein the spacer sequence binds to a target nucleic acid.

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

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

4. The system of claim 3 , wherein the direct repeat sequence comprises the nucleotide sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10.

5. 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.

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

7. The system of claim 6 , wherein the spacer sequence comprises between 24 and 38 nucleotides in length.

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

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

10. The system of claim 9 , wherein the PAM sequence comprises a nucleotide sequence set forth as 5′-TTY-3′, wherein Y is C or T.

11. The system of claim 1 , wherein the CRISPR-Cas effector protein cleaves the target nucleic acid.

12. 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.

13. 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.

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

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

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

17. The system of claim 16 , 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.

18. 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.

19. A cell comprising the system of claim 1 .

20. 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.

Assignees

• Arbor Biotechnologies Inc

Inventors

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

Classifications

• C12N2310/20

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

• C12N2310/531

  Stem-loop; Hairpin · CPC title

• C12N2800/80

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

• C12N9/22 Primary

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

• C12N15/102

  Mutagenizing nucleic acids · CPC title