CRISPR/Cas9编辑绒山羊FGF5基因细胞株的建立

拟利用CRISPR/Cas9技术建立编辑FGF5基因的绒山羊细胞株。在FGF5基因的第一外显子设计靶点并合成gRNA靶点引物，构建2个编辑FGF5基因的Cas/gRNA真核表达质粒载体。电穿孔法转染绒山羊成纤维细胞后T7核酸内切酶（T7E1）检测载体活性，选择活性最高的载体转染细胞，单细胞接种并扩繁，提取基因组DNA，PCR及测序鉴定。经测序分析共获得20个FGF5基因敲除细胞株（包括FGF5^+/-和FGF5^-/-），总突变率为14.81%。双敲除突变细胞株可作为供体细胞进行重构胚构建，为创造高产绒性状的FGF5基因编辑绒山羊奠定基础。

Establishment of CRISPR/Cas9-edited FGF5 Cell Strains in Cashmere Goat

To establish CRISPR/Cas9-edited FGF5 cell strains, we designed gRNA targeted sites around the first extra of the FGF5 gene and constructed two vectors by Cas/gRNA plasmid construction kit weredesigned. vectors into cashmere goat fibroblasts by electroporation respectively was transfected. T7 endonuclease 1 (T7E1) was used for the detection of mutation efficiency. The best vector was transfected into cashmere goat fetal fibroblasts and all the monoclones was cultured. Then all the cell colonies by sequencing were idenfified. Sequencing results demonstrated that CRISPR/Cas9 was available for FGF5 gene edited and 20 FGF5^+/- and FGF5^-/- cell colonies were obtained, and the effiency was 14.81%. The double mutated cell colonies could be used as donor cells to construct reconstructed embryos which provide the possibility in production of FGF5 edited cashmere goat in the future.