Preparetion Of KRTAP13-1 Knockout Cashmere Goats By CRISPR/Cas9 System

Inner Mongolia White Cashmere Goat is the highest cashmere yield and the best cashmere fiber quality goat breed in the world.Its cashmere output accounts for more than half of China's cashmere output,accounting for 1/3 of the world's cashmere output.Cashmere,as the most important economic component of Inner Mongolia White Cashmere Goats,is a kind of rare and precious animal resources and enjoys a high reputation in the world.However,due to the large demand of international cashmere market,cross-breeding for the purpose of increasing yield have been carried out in various producing areas,which has dramatically reduced the quality of cashmere and seriously damaged the genetic resources.Traditional biological breeding also faces the problems of few stock lines and long breeding cycle.The application of gene editing technology provides a new direction for large animal breeding.Keratin-associated protein(KAP)is an important component of keratin,which has an important influence on the development and structure of skin derivatives such as hair.The keratin-associated protein gene(KRTAP)13-1,a regulatory gene of KAP13-1,was found to be involved in the early formation of hair follicles and affect the diameter of cashmere.In this study,KRTAP13-1 gene knockout cashmere goats were prepared by CRISPR/Cas9 system to investigate the effects of KRTAP13-1 gene on hair follicle development and fiber diameter,which provided theoretical and research basis for breeding new superfine cashmere goats.1.Vector ConstructionFour pairs of gRNA were designed in the coding sequence(CDS)region of KRTAP13-1 gene,and Cas9/gRNA co-expression vectors were constructed.After Surveyor detection and locus analysis,Cas9/gRNA co-expression vector No.3 was selected for this research.2.Screening of Monoclonal Cell LinesIn this experiment,the fetal fibroblasts of cashmere goats after 48 hours of transfection were separated into 96-well plates for single cell culture by flow cytometry and oral pipette.A total of 100 monoclonal cell lines were obtained,17 KRTAP13-1 single allele targeted cell lines was obtained and the targeting efficiency was 17%;3 KRTAP13-1 double allele targeted cell lines was obtained and the targeting efficiency was 3%.3.Preparation of KRTAP13-1 gene targeting Cashmere GoatsIn this experiment,979 ovaries were collected from local goat slaughterhouse and 5542 oocytes were obtained by mechanical cutting.2971 oocytes were matured after in vitro culture,and the maturation rate was 53.6%.After cloning and fusion,1928 reconstructed embryos were formed,of which 1154 developed cleavage stage,with a cleavage rate of 59.8%.The reconstructed embryos were transferred into oviduct of 170 recipient goats,7 kids were obtained after pregnancy,and 3 of them were identified as KRTAP13-1 gene knockout goats.