Abstract: This study was aimed to use homologous recombination mediated by Tild-CRISPR/cas9 system to acquire homozygous hornless cell lines. sgRNA were designed for Pc target site controlling the polledness in cattle, and its mutation efficiency was detected by T7E1 digestion and sequencing. pUC57 vector was used as the skeleton to produce homologous recombinant fragment through connecting the Pc fragment and homology length of 1 600 bp (800 bp homology on each arm), then the homologous recombinant fragment was obtained by double enzyme digestion and sgRNA were co-transfected into the fibroblasts of the auricular margin in Holstein bulls, the single cell clone was screened and identified. The results showed that six sgRNAs_(1-6) were successfully constructed and sgRNA₁ with the highest mutation efficiency of 32.6% was obtained. The homologous recombination fragment of 1 901 bp was successfully obtained. A total of 147 single cell clones were obtained, and 8 single cell clones had correct single allele Pc site insertion, and 1 single cell clone had corrected double allele Pc site insertion. The results of foreign gene residue test showed that there was no Cas9 gene residue in single cell clone, which could be used as the nuclear donor cells for subsequent cloning and production of excellent hornless homozygous embryos. This study successfully used Tild-CRISPR/Cas9 site-specific editing method to obtain the ear margin fibroblast cell line without exogenous Cas9 gene residue with homozygous insertion at PC site, which provided a good material reserve and technical platform for the breeding of excellent bulls and the research of main functional genes in the future.

Key words: Holstein dairy cows; hornless; Pc site; Tild-CRISPR/Cas9; homologous recombinant vector