Abstract: 【Objective】 The objective was to explore the feasibility of knockout of ev21 gene in slow feathering chicken fibroblasts for the endogenous retroviruses purification of chicken flocks, and lay a foundation for rapid breeding of slow feathering chicken lines without ev21 gene.【Method】 Based on the characteristics of ev21 gene sequence(KY235336), two sgRNAs were designed at each of its 5' and 3' ends, respectively, for the construction of four different sgRNA targeting plasmids, and the sgRNAs with higher efficiency at the 5' and 3' ends were screened. Then the ev21 gene was cut based on CRISPR/Cas9 gene editing technology and the ev21 gene was replaced by a DNA fragment of red fluorescent protein(mCherry) (CAG-mCherry) by homologous recombination to achieve targeted knockout of the ev21 gene of endogenous leukemia virus in slow feathering chicken fibroblasts.【Result】 The results showed that the ev21 gene could be detected in slow feathering chicken fibroblasts, and the four constructed sgRNAs(sgRNA1-sgRNA4) could be successfully inserted into the corresponding targeting plasmids. After puromycin screening and T7E1 digestion assay, it was found that slow feathering chicken fibroblasts had different degrees of death after transfection with the four different sgRNAs, among which the knockdown efficiency of sgRNA1 and sgRNA3 was higher. Donor plasmids containing the left and right homology arms and expressing mCherry were also constructed with reference to the homologous site. All 293T cells were able to express mCherry after 12 h transfection with the donor plasmid. Cells were found to consistently express mCherry when slow feathering chicken fibroblasts were co-transfected with sgRNA1 and sgRNA3 targeting plasmids and donor plasmids. Red fluorescent positive fibroblasts were collected by flow cytometry at day 30 after transfection, and their total DNA was extracted for PCR identification and gene sequencing, which showed that the target fragment(CAG-mCherry) was inserted in the red fluorescent positive fibroblasts, and therefore the knockdown of ev21 gene could be achieved by insertion substitution.【Conclusion】 The gene knockout method based on CRISPR/Cas9 gene editing technology can successfully knock out the ev21 gene of endogenous leukemia virus in slow feathering chicken fibroblasts, and provide technical support for breeding slow feathering chicken strains without ev21 gene.