US 11,180,763 B2 · CRISPR/Cas9 vector combination and application thereof in gene knockout

Abstract

Provided is an SgRNA combination, comprising an SgRNA specifically targeting the GGTA1 gene, an SgRNA specifically targeting the CMAH gene and an SgRNA specifically targeting the β4GalNT2 gene. Also provided is a CRISPR/Cas9 vector combination, comprising a GGTA1-CRISPR/Cas9 vector, a CMAH-CRISPR/Cas9 vector and a β4GalNT2-CRISPR/Cas9 vector. Also provided is an applicaton of the CRISPR/Cas9 vector combination in knocking out the GGTA1 gene, the CMAH gene and the β4GalNT2 gene. The knockout rates of the three genes with the specifically targeted SgRNA sequences are respectively 56%, 63%, and 41%. A three genes knockoutpig can be obtained, wherein the three genes related to immune rejectionare knocked out, and heart valves of said pig can be acquired.

Claims8

Dependent claims

A single guide RNA (SgRNA) combination, comprising an SgRNA specifically targeting a α-1,3-galactosyltransferase (GGTA1) gene, an SgRNA specifically targeting a cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene and an SgRNA specifically targeting a β1,4-N-acetyl galactosaminyl transferase 2 (β4GalNT2) gene;

wherein, the SgRNA specifically targeting the GGTA1 gene comprises the nucleotide sequence as set forth in SEQ ID No:1, the SgRNA specifically targeting the CMAH gene comprises the nucleotide sequence as set forth in SEQ ID No:2, and the SgRNA specifically targeting the β4GalNT2 gene comprises the nucleotide sequence as set forth in SEQ ID No:3.

A vector combination

comprising a GGTA1 vector, a CMAH vector and a β4GalNT2 vector;
wherein the GGTA1 vector comprises a SgRNA specifically targeting a GGTA1 gene, and the SgRNA comprises the nucleotide sequence as set forth in SEQ ID No:1;
the CMAH vector comprises a comprises a SgRNA specifically targeting a CMAH gene, and the SgRNA comprises the nucleotide sequence as set forth in SEQ ID No:2, and
the β4GalNT2 vector comprises a SgRNA specifically targeting a β4GalNT2 gene, and the SgRNA comprises the nucleotide sequence as set forth in SEQ ID No:3.

1 more element

Dependencies are inferred from the claim text.

CRISPR/Cas9 vector combination and application thereof in gene knockout

Life of this patent

CRISPR/Cas9 vector combination and application thereof in gene knockout

Life of this patent

Abstract

Claims8