Construction Of FGF5 Gene Knockout Rabbit By CRISPR/Cas9

Hair is skin appendages and its growth as well as morphology are generally regulated by the hair cycle in mammals.The periodic hair cycle can be typically defined as three phases:anagen,the stage during which a new hair is produced;catagen,the stage during which hair elongation ceases;telogen,the stage during which the follicle is at rest;and exogen,the stage during which the hair falls actively.In addition,the hair cycle is thought to be composed of one or several activator/inhibitor pairs acting to regulate key cycle phase transitions at set thresholds of their activities.Fibroblast Growth Factor 5?FGF5?is a hair cycle regulator and a hereditary long hair phenotype has been reported when FGF5 is mutated in humans and other species.However,there has been no such report investigating the alternation of hair follicle cycle inducing by disruption of FGF5 in rabbits.In this study,two FGF5-sgRNAs were designed based on rabbit genome sequence and the mix of Cas9 mRNA and FGF5-sgRNA were injected into cytoplasm of rabbit embryo.Then,the injected embryos were transferred to pseudo pregnant recipient rabbits.Two FGF5^-/-rabbit was identified by determination of genomic DNA in F0generation and more examples were generated by mating with FGF5^-/-rabbit of F0generation.FGF5^-/-and WT rabbits of F2 generation were selected for determination of genotype by genome analysis.In addition,the disruption of FGF5 in FGF5^-/-rabbit was confirmed by IHC?immunohistochemistry?,RT-QPCR and WB.The systemic effect of long hair due to rapid growth rate could be detected in FGF5^-/-rabbit.Further,female rabbits are more sensitive to long hair meditating by disruption of FGF5.Though no significant difference in total hair follicle number were discovered,higher ratio of hair follicle cluster,diameter of hair follicle and ratio of secondary/primary hair follicle were detected in Hematoxylin and eosin?H&E?staining of FGF5^-/-rabbit compared to WT rabbit.No significant difference of hair structure and shaft while a decreased diameter and width of down and guard hair squama could be detected in SEM?scanning electron microscope?results.Further,the decreased expression level of BPM2/4 pathway and increased expression level of VERSICAN pathway confirmed that the disruption of FGF5,due to prolonging of anagen,results in long hair in rabbits.Taken together,FGF5^-/-rabbit with long hair genotype offers a high effective way for genetic improvement in agricultural and an ideal model for studying the mechanism of long hair and hair cycle in the future.