Generation of Buffalo Mammary Epithelial Cells with Targeted Knockout of 18s Rdna by a CRISPR/Cas9 Adenovirus System

In recent years, CRISPR/Cas9 has rapidly become one of the most promising genome editing tools because it is simple and easy to use and cost effective. However, the large size of Cas9 sequences limits its application in clinically promising vectors and it also impacts non-viral transfection. In this study, CRISPR/Cas9 adenovirus vectors that target the buffalo 18s rDNA gene were constructed, transfected into 293 cells for adenovirus packaging, and the adenovirus was used to knockout the 18s rDNA gene in buffalo mammary epithelial cells. The results demonstrated that the CRISPR/Cas9 adenovirus vectors for the buffalo 18s rDNA gene could efficiently target the sites as revealed by the fluorescence reporter system. After amplification, the adenovirus titer of Sn458-18s1 and Sn458-18s2 reached 1.03 × 10 9 PFU/mL and 1.05 × 10 9 PFU/mL, respectively. For buffalo mammary epithelial cell infection, the efficiency was 100% when the multiplicity of infection (MOI) ≥ 100 PFU/mL. There were 9 mutational clones found in the 20 clones, and the gene mutagenesis rate reached 45%. Of these, 2 clones were 35-bp deleted and 7 clones were 12-bp deleted. These results suggested that the adenovirus system overcame the low transfection efficiency of the buffalo mammary epithelial cells associated with using lipid-based methods or electroporation. Moreover, we preliminary developed an efficient technique for multiple-locus gene targeting at repeated sequences of the buffalo genome.