Generation Of MSTN Gene Knock-Out And Fat-1 Gene Knock-In Goat Via CRISPER/Cas9

The aims of genetic manipulation are to enhance agriculture by modifying domestic animal, to maximize yields and value.MSTN is known as a negative regulator of muscle development, mutations in the gene can lead to significantly increases in muscle development. Fat-1 gene encoding ω-3 PUFAs desaturase, can convert polyunsaturated fatty acids from ω-6 to ω-3, the increase of fat-1 gene is good for improving the content of co-3 in animals. Our works is to construct gRNA expression vectors and new plasmid vectors for fat-1 gene targeting in MSTN gene locus, which enable knock-out MSTN gene and knock-in fat-1 gene at the same time. GEFs were transfected with those vectors, along with selecting the best co-electroporation conditions by electroporation. Our method involved generating gene modified GEFs and using SCNT to generate transgenic goats which improve yield and quality of mutton.1. Vector constructionTwo gRNA Vectors has been constructed successfully at MSTN of exon 1, the mutation efficiency of gRNA2 is higher than gRNA1 through the survery enzyme mutation detection, so gRNA2 is used as follow-up experiments; Sequencing confirmed targeting gene fragment was obtained and the fat-1 gene targeting vector in MSTN gene locus is constructed successfully.2. Production of gene-modified monoclonal cellsTo screen the monoclonal cell lines by the methods of glass tube and flow cytometer, hCas9 vector, gRNA and donor plasmid were transfected into GFFs via electroporation.Out of 156 monoclones 40 strains homologous recombinant monoclones were obtained by both PCR and sequencing analysis. The efficiency of gene konck-in was 25.64%.3. Generation of transgenic goats249 cloned embryos were produced from genetically modified monoclonal cells through nuclear transfer.134 cloned embryos were transferred into 56 recipient female goats. Recently, a lamb was born after embryos transfer. The result of PCR indicated that the fat-1 gene had been integrated into the MSTN locus of goat genome.