Application Of CRISPR/Cas9 For Myostatin Gene Knockout Cell Lines

Gene editing technology has been developed rapidly in recent years. People can knock out, insert and site-directed mutate gene through gene editing technology to study the gene function and regulation elements, which is different from traditional cloning techniques. Great achievements in the technical aspects of gene pharmaceuticals, diagnosis, therapy through gene editing technology has been achieved, which would change the face of human life greatly.CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) is a promising gene modification tools, as the third-generation artificial endonuclease technology. CRISPR / Cas system is an adaptive immune defenses in the formation of long-term evolution of bacteria and archaea, which could be used to fight against invading viruses and exogenous DNA. The derivative CRISPR / Cas9 technology shows lower cost, lower toxicity, wider application, shorter operating cycle, etc., compared to the previous ZFNs(Zinc-finger nucleases) and Talens(transcription activator-like effector nuclease) technology. It would bring a breakthrough revolution for targeted genomic transformation, regulation and applications. However, bovine cells have not been knocked out without screened marker gene by CRISPR / Cas9 technology currently.Myostatin,(MSTN), also known as GDF-8, belong to TGF-β superfamily. It could regulate the muscle growth negatively through the regulation of the number, size and growth rate of muscle cells. The deletion or inactivation of mammalian MSTN gene leads to larger muscle fiber diameter or increased number of muscle fibers, resulting in excessive muscle growth. Natural mutation or gene editing can make the anti-born functional disorders of MSTN gene, leading to the animals appear trait of "double-muscled". The research of MSTN gene has great significance for the production of animal meat and has been studied extensively. Therefore, we can obtain the "double-muscled" beef through genetic editing techniques, which would have a very important value and broad application.In this study, we designed the target sites of bovine MSTN gene exon sequences randomly by different expression vectors of CRISPR / Cas9 system. Target site activity was detected through T7 endonuclease I(T7EI) without adoption of the traditional method of SSA. The expression vector of highest knockout efficiency and target site was selected from the designed target sites and vectors through T7 endonuclease I in the condition of in the absence of screening marker. We knocked out gene for Luxi(LXHN) fetal fibroblast cells, prepared the single cell cloning by limiting dilution, identified cell clones by PCR sequencing and T7 EI in order to obtain MSTN gene knockout cell lines and prepare provided materials for transgenic animals.Detected the effect of double-target on mutation efficiency simply in knockout experiment at the same time.The main research results are as follows: 1.Construction of CRISPR/Cas9 targeting vectorThe target site of bovine MSTN gene the second exon are ps Pg RNA-M-A、p X330-M-A;the target site of bovine MSTN gene the third exon are ps Pg RNA-M-B、p X330-M-B、ps Pg RNA-M-X and ps Pg RNA-M-Y. 2.Mutation efficiency of CRISPR/Cas9 targeting vectorMutation efficiency using T7 EI enzyme assay at different target sites and the highest mutation efficiency is 36.67%. 3.Preparation of single cloning cells by limiting dilution and screening MSTN mutation cell lines53 clones were obtained.We screened 5 MSTN gene knockout cell clones(MSTN-/-). 4.Double-target site on mutation efficiencyThe third exon three target sites of two two combinations for mutations in efficiency which prove that the double-target mutation efficiency is on the targets’ distance.