Establishment Of CRISPR/Cas9-edited FGF5 Cell Strains In Cashmere Goat

The quality and quantity of cashmere is associated with growth cycle of secondary follicle in Inner Mongolia goat.Fibroblast growth factor 5(FGF5),the member of the FGFs gene family factor,plays an important role in regulating the hair growth cycle,the fact is demonstrated by many studies.The mutational FGF5 gene can lead to the extension of hair growth cycle in the growth period,resulting in increasing the length of the hair.This study was aim to establish gRNA-CRISPR/Cas9 gene editing plasmid,transfect vector into the fetal fibroblasts of cashmere goat by electroporation method,screen and cuture all the cell strains.Then all the cell colonies were identified by sequencing.Sequencing results demonstrated that CRISPR/Cas9 was available for FGF5 gene edited and FGF5-/-cell colonies were obtained.The double mutated cell colonies could be used as donor cells to construct reconstructed embryos,which lays the basic foundation in production of FGF5 edited cashmere goat in the future.1.We designed two gRNA targeted sites around the first exon of FGF5 gene and construct two vectors named pCas9-FGF5-1 and pCas9-FGF5-2.The sequencing results showed that the vectors were successfully constructed.T7 endonuclease 1 enzyme identification and SSA activity detection were used for measuring mutation efficiency.Two vectors were both available.pCS7-FGF5-2 could be used for the following research.2.pCS7-FGF5-2 was transfected into cashmere goat fetal fibroblasts with NEPA21 electricity transfection.All the monoclones were cultured and expanded.We identified all the cell colonies by sequencing.The results showed that 20 FGF5 colonies were succeed.After T-A sequencing identification,there were three cell strains inserted the single base in the target area,and one cell strain with 23 bases deletion in one allele,missing 12 bases deletion in the other.The effiency was 14.81%(including FGF5+/-and FGF5-/-),and the mutation effiency of FGF5-/-is 2.96%.We obtain the FGF5-/-cell colonies which provide the possibility in producting transgenic cashmere goats.