Engineering of systems, methods and optimized guide compositions for sequence manipulation

Patent metadata

Field	Value
Publication number	US-2025250553-A1
Application number	US-202519025692-A
Country	US
Kind code	A1
Filing date	Jan 16, 2025
Priority date	Dec 12, 2012
Publication date	Aug 7, 2025
Grant date	—

Abstract

The invention provides for systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR-Cas system.

First claim

1 . (canceled)

2 . An engineered CRISPR-Cas system chimeric RNA comprising NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG CUAGUCCGUUAUCA.

3 . The engineered CRISPR-Cas system chimeric RNA of claim 2 , wherein NNNNNNNNNNNNNNNNNNNN is a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell adjacent to a protospacer adjacent motif (PAM).

4 . The engineered CRISPR-Cas system chimeric RNA of claim 3 , wherein the PAM is NGG.

5 . The engineered CRISPR-Cas system chimeric RNA of claim 2 , further comprising a poly-U sequence.

6 . The engineered CRISPR-Cas system chimeric RNA of claim 2 , wherein the RNA sequence is encoded by SEQ ID NO:26.

7 . The engineered CRISPR-Cas system chimeric RNA of claim 2 , comprising one or more modified nucleotides.

8 . The engineered CRISPR-Cas system chimeric RNA of claim 2 , comprising one or more methylated nucleotides or nucleotide analogs.

9 . An engineered CRISPR-Cas system chimeric RNA comprising, from 5′ to 3′:
(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell adjacent to a protospacer adjacent motif (PAM),
(b) a tracr-mate sequence, and
(c) a tracr sequence comprising at least 40 nucleotides in length,
wherein the tracr-mate sequence is capable of hybridizing to the tracr sequence, and wherein the chimeric RNA is capable of forming a CRISPR complex with S. pyogenes Cas9 and directs sequence-specific binding of the CRISPR complex to the target sequence adjacent to the PAM in the eukaryotic cell.

10 . The engineered CRISPR-Cas system chimeric RNA of claim 9 , wherein tracr sequence comprises at least 50 nucleotides in length.

11 . The engineered CRISPR-Cas system chimeric RNA of claim 9 , wherein guide sequence comprises 15-25 nucleotides in length.

12 . The engineered CRISPR-Cas system chimeric RNA of claim 9 , wherein the PAM is NGG.

13 . The engineered CRISPR-Cas system chimeric RNA of claim 9 , further comprising a poly-U sequence.

14 . The engineered CRISPR-Cas system chimeric RNA of claim 9 , comprising one or more modified nucleotides.

15 . The engineered CRISPR-Cas system chimeric RNA of claim 9 , comprising one or more methylated nucleotides or nucleotide analogs.

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College

Inventors

• Zhang Feng
• Cong Le
• Hsu Patrick
• Ran Fei

Classifications

• C12N2810/50

  Vectors comprising as targeting moiety peptide derived from defined protein · CPC title

• C12N2800/10

  Plasmid DNA · CPC title

• C12N15/907

  in mammalian cells · CPC title

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title

• C12N15/86

  Viral vectors · CPC title