Abstract：In the cattle industry, the appendages such as horns have become undesirable because they are dangerous to the breeders and animals. The polled (hornless) condition in cattle has existed since domestication, and it has been selected because of its economic importance and ease of management. A single dominant mutation is considered to be the cause of polled phenotype, called POLLED locus. But in realistic production, there are few cattle with this site. In order to produce polled phenotype more efficiently, the present study used CRISPR/Cas9 gene editing technique to mediate knock-in of P202ID genotype in POLLED locus of horned Mongolian cattle (Bos taurus). First, four pairs of single guide RNA (sgRNA) targeting to bovine POLLED site, named as sgRNA1~4, were designed, and inserted into the pCas-Guide-EF1α-GFP plasmid. The results of surveyor assay showed that the mutant efficiency for 4 vectors amounted to 24%, 26%, 55%, and 17%, respectively. pCas-Guide-EF1α-GFP-sgRNA3 vector had the highest activity in the vectors. The high active vector and targeting carrier containing P202ID sequence were co-transfected into Mongolian cattle fetal fibroblasts by electrotransfection. The positive single cells were separated by flow cytometry and further cultured into monoclonal cells. By PCR amplification and sequencing, 4 strains of heterologous recombinant monoclonal cell lines were obtained, which could be used as a donor cell for subsequent somatic cell nuclear transfer (SCNT). This study could provide experimental materials and technical support for cultivation of POLLED cattle with SCNT, and contribute important materials for the understanding of the mechanism of horn origin and development.
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