CRISPR DNA targeting enzymes and systems

Patent metadata

Field	Value
Publication number	US-12460190-B2
Application number	US-202017641523-A
Country	US
Kind code	B2
Filing date	Sep 9, 2020
Priority date	Sep 9, 2019
Publication date	Nov 4, 2025
Grant date	Nov 4, 2025

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 capable of hybridizing to a target nucleic acid in a eukaryotic cell; 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: 10, 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 direct repeat sequence comprises a nucleotide sequence with at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 291.

3 . The system of claim 2 , wherein the direct repeat sequence comprises the nucleotide sequence set forth in SEQ ID NO: 291.

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

5 . The system of claim 4 , wherein the spacer sequence comprises between 30 and 51 nucleotides in length.

6 . The system of claim 4 , wherein the spacer sequence comprises between 15 and 23 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′-RTTR-3′, 5′-ATTR-3′, or 5′-RTTG-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 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 cell comprising the system of claim 1 .

18 . The cell of claim 17 , wherein the cell is a eukaryotic cell.

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

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

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

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

23 . The method of claim 21 , wherein binding the system to the target nucleic acid results in cleavage of the target nucleic acid.

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

25 . The system of claim 1 , wherein the CRISPR-Cas effector protein has at least 99% identity to SEQ ID NO: 10.

Assignees

• Arbor Biotechnologies Inc

Inventors

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

Classifications

• C12Q1/6823

  Release of bound markers · CPC title

• C12N2800/80

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

• C12N2800/10

  Plasmid DNA · CPC title

• C12N15/85

  for animal cells · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title