	US 12,369,569 B1
	Method for generating genome-edited chickens
Ning Yang, Beijing (CN); Xiqiong Wang, Beijing (CN); and Congjiao Sun, Beijing (CN)
Assigned to CHINA AGRICULTURAL UNIVERSITY, Beijing (CN)
Filed by CHINA AGRICULTURAL UNIVERSITY, Beijing (CN)
Filed on Dec. 17, 2024, as Appl. No. 18/984,398.
Application 18/984,398 is a continuation of application No. PCT/CN2024/106891, filed on Jul. 23, 2024.
Claims priority of application No. 202410931448.9 (CN), filed on Jul. 12, 2024.
Int. Cl. A01K 67/0275 (2024.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01)

CPC A01K 67/0275 (2013.01) [C12N 9/22 (2013.01); C12N 15/111 (2013.01); A01K 2227/30 (2013.01); C12N 2310/20 (2017.05)]

10 Claims

1. A method for generating a gene-edited chicken with a IHH gene knockout, comprising the following steps:

injecting a gene editing reagent into ovarian medulla of a hen 10 days to 15 days before the hen lays egg such that a G₋₁hen is obtained,

artificially inseminating the G₋₁hen such that fertilized eggs are obtained, and artificially hatching the fertilized eggs such that a G₀population is obtained; and

detecting gene editing results in the G0 population to identify the gene-edited chicken;

wherein the gene editing reagent comprises a Cas9 protein, at least two guide RNA (gRNA), and a buffer, and the Cas9 protein and each gRNA of the at least two gRNA form a ribonucleoprotein (RNP);

wherein a combined editing efficiency of the at least two gRNA is detected using a chicken tool cell line before the gene editing reagent is injected, and wherein the at least two gRNA have a combined editing efficiency greater than or equal to 45%;

wherein each gRNA of the at least two gRNA target sequences are in the IHH gene; and wherein the at least two gRNA comprise the nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 3.