CRISPR RNA targeting enzymes and systems and uses thereof

Patent metadata

Field	Value
Publication number	US-12460203-B2
Application number	US-202117521617-A
Country	US
Kind code	B2
Filing date	Nov 8, 2021
Priority date	Jun 30, 2017
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 a nucleic acid.

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 comprising a direct repeat sequence of SEQ ID NO: 65 and a spacer sequence capable of hybridizing to a target nucleic acid in a eukaryotic cell; and (b) a nucleic acid encoding a CRISPR-Cas effector protein, or the CRISPR-Cas effector protein, 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 consists of an RNA transcript of the nucleotide sequence set forth in SEQ ID NO: 65.

3 . The system of claim 1 , wherein the RNA guide comprises a truncated direct repeat sequence.

4 . The system of claim 1 , wherein the spacer sequence comprises between 15 and 42 nucleotides in length.

5 . The system of claim 1 , wherein the CRISPR-Cas effector protein comprises at least two HEPN domains.

6 . The system of claim 1 , wherein the CRISPR-Cas effector protein comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 26.

7 . The system of claim 6 , wherein the CRISPR-Cas effector protein comprises the amino acid sequence set forth in SEQ ID NO: 26.

8 . The system of claim 1 , wherein the target nucleic acid is an RNA molecule.

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

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, an RNA methyltransferase, an RNA demethylase, a splicing modifier, a localization factor, or a translation modification 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 RNA guide is operably linked to a promoter.

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

15 . The system of claim 1 , wherein the nucleic acid encoding the RNA guide is in a vector.

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 1 , wherein the nucleic acid encoding the RNA guide and the nucleic acid encoding the CRISPR-Cas effector protein are in a vector.

18 . The system of claim 15 , 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.

19 . 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.

20 . A cell comprising the system of claim 1 .

21 . 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

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

Classifications

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title

• C07K14/195

  from bacteria · CPC title

• C12N9/22

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

• C12N2310/20

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

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title