Modified Cas9 system having a dominant negative effector on non-homologous end-joining fused thereto and its use for improved gene editing

Patent metadata

Field	Value
Publication number	US-12492389-B2
Application number	US-202118042763-A
Country	US
Kind code	B2
Filing date	Jul 26, 2021
Priority date	Sep 4, 2020
Publication date	Dec 9, 2025
Grant date	Dec 9, 2025

Abstract

The present invention relates to modified Cas9 nuclease comprising a substantial part of a Cas9 nuclease and fused thereto at least one substantial part of a dominant negative effector on non-homologous end-joining selected from the group consisting of RNF168, 53BP1, Ku80 and DNA-PK which compete with NHEJ promoting factors and CtIP.

First claim

The invention claimed is:

1 . A fusion protein comprising (1) wild type Streptococcus pyogenes Cas9 nuclease, (2) full-length, unmodified human C-terminal binding protein interacting protein (CtIP) and (3) the polypeptide of SEQ ID NO: 2.

2 . A nucleic acid coding for the fusion protein according to claim 1 .

3 . The nucleic acid according to claim 2 characterized in that said nucleic acid is a DNA sequence.

4 . The nucleic acid according to claim 2 characterized in that said nucleic acid is an mRNA sequence.

5 . A vector for the transfection of target cells characterized in that said vector comprises a nucleic acid according to claim 2 .

6 . The vector according to claim 5 characterized in that said vector is a plasmid.

7 . The vector according to claim 5 , wherein said vector is a recombinant viral vector.

8 . A method for editing genomic sequences via homology directed repair using the fusion protein of claim 1 , said method comprising the steps of:
(i) introducing said fusion protein and a guide RNA (gRNA) in a human cell, whereby said fusion protein generates a double stranded break at a specific site in the genome of said human cell to yield two cleaved genomic strands; and (ii) transferring into said cell a complementary donor template designed to rejoin the cleaved genomic strands by means of homology directed repair.

9 . The vector according to claim 7 , wherein said recombinant viral vector is selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, and retroviral vectors.

10 . An engineered CRISPR-Cas9 system for editing genomic sequences, said system comprising (1) the fusion protein of claim 1 and (2) a chimeric single guide RNA (gRNA) in the form of a single strand RNA molecule that consists of (a) 20 nucleotides complementary to a selected genomic sequence and (b) a remaining portion that folds into a tridimensional structure capable of forming a complex with the wild type Streptococcus pyogenes Cas9 nuclease portion of said fusion protein.

11 . A method for editing genomic sequences via homology directed repair, said method comprising the step of:
a. delivering into a human cell the engineered CRISPR-Cas9 system of claim 10 , whereby said complementary nucleotides of said gRNA hybridize to a corresponding complementary section of said cell's genome while said remaining gRNA portion forms a tridimensional complex with the wild type Streptococcus pyogenes Cas9 nuclease portion of said fusion protein capable of cleaving said genome into two genomic strands; b. generating a double stranded break in said cell's genome at the site of hybridization; and c. transferring into said cell a complementary donor template designed to rejoin the cleaved genomic strands at the site of hybridization by means of homology directed repair.

12 . The method of claim 11 , wherein said human cell comprises primary human T lymphocytes or hematopoietic stem cells (HSC).

Assignees

• Univ Freiburg Albert Ludwigs

Inventors

• Mussolino Claudio
• Cathomen Toni
• Cornu Tatjana
• Carusillo Antonio

Classifications

• C12N15/907

  in mammalian cells · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12N2310/20

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

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C07K19/00

  Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes} · CPC title