One-step Generation Of FGF5 Gene Knockout Sheep Via The CRISPR/Cas9 System

The wool is an important agricultural product in global economy.Wool length is one of the most valuable wool quality traits in sheep breeding.It has been verified that fibroblast growth factor 5(FGF5)is an inhibitor of hair elongation,as FGF5-null mouse exhibit an extremely long hair phenotype.The underlying mechanism clarified that silencing the expression of FGF5 gene prolonged the growth phase of the hair cycle,resulting in a phenotype of extremely long hair.Recently,a novel and efficient genome editing tool based on the clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated(Cas)system 9 has been booming.The CRISPR/Cas system injected into a 1-cell embryo that was efficiently generate genetically engineered model animals and livestock.In this study,we successfully generated FGF5 knockout sheep using the CRISPR/Cas system by direct 1-step cytoplasmic injection of Cas9 mRNA and sgRNA(Single Guide RNA)into zygotes.To promote the codon adaptation,the Cas9 sequence from Streptococcus pyogenes SF370 was codon optimized for sheep.Three sgRNAs designed for target the FGF5 gene in sheep genome.Then,the sequence of optimized Cas9 and sgRNA were cloned and constructed the eukaryotic expression plasmid of U6-CMV-Cas9-H1.Cas9 mRNA and FGF5 sgRNAs were obtained by In vitro transcription respectively.The Cas9 mRNA and sgRNAs injected into cytoplasm of 1-cell embryo cell.Immunofluorescence and RT-PCR confirmed that expression of Cas9 could be detected within 8-cells embryos and sgRNA disappeared until 16-cells embryos.To generate the FGF5 knockout sheep,in total,158 zygotes were collected and 155 1-cell embryos one-step microinjected with Cas9 mRNA and sgRNA.One hundred and fifty five embryos injected transferred into 53 surrogate females.Fourteen pregnant ewe were established,and borned 18 lambs,inclulding 4 twins.Three genome editing lambs were identified by PCR and sequencing.Sequencing analysis confirmed that mutations in FGF5 gene existed in all germ lines of three founders:besides the intact sequence,three kinds of deletions in FGF5 gene(including 5,13,and 33 bp)detected.The changes in the primary and senior structure of the FGF5 protein due to the theree deletions in founders suggested that the biological function of FGF5 protein was lost.In addition,the expression level of intact FGF5 mRNA in heterozygous individuals decreased compared with the wild types(p<0.01).Functionally,we discovered that wool length in founders was significantly longer than in wild types(p<0.05).Collectively,the FGF5 knockout sheep with the longer wool length phenotype will provide an efficient way for fast genetic improvement of sheep breeding and promote the development of wool industry.