利用Tild-CRISPR/Cas9定点编辑荷斯坦种公牛的无角Pc位点

Targeted Editing of Hornless Pc Site in Holstein Bulls Using Tild-CRISPR/Cas9

试验旨在利用Tild-CRISPR/Cas9系统介导的同源重组技术制备纯合无角种公牛细胞系。针对控制无角性状的Pc靶位点设计sgRNA,T7E1酶切和测序检测突变效率;以pUC57为骨架连接Pc片段及其两侧约800 bp的同源序列左臂(L)和右臂(R),双酶切获得同源重组片段后与sgRNA共同转染至荷斯坦种公牛耳缘成纤维细胞中,筛选获得单细胞克隆并鉴定。结果显示,试验成功构建了6个sgRNA(1-6),并筛选获得了突变效率最高的sgRNA1,其突变效率为32.6%;成功获得了长度为1901 bp基因序列完全正确的同源重组片段;共获得147个单细胞克隆,其中8个单细胞克隆发生正确的单等位Pc位点插入,1个单细胞克隆发生正确的双等位Pc位点插入。外源基因残留检测结果显示,单细胞克隆没有Cas9基因残留,可作为后续克隆生产优秀无角纯合胚胎的核供体细胞。本研究成功利用Tild-CRISPR/Cas9定点编辑的方法获得了Pc位点纯合插入的无外源Cas9基因残留的无角牛耳缘成纤维细胞系,为将来优秀无角体细胞克隆种公牛的培育以及主要功能基因研究提供了良好的材料储备和技术平台。

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 1600 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 1901 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.