Knock Out MSTN Gene Of Arbas Cashmere Goat By CRISPR-Cas9

Myostatin (also known as growth differentiation factor 8, abbreviated GDF-8) is an important negative regulator of skeletal muscle growth. Studies have shown that the mutation in the MSTN gene to cause myostatin-related muscle hypertrophy including promoting of muscle growth and the change of the composition of red and white muscle and the increasing of muscle mass. CRISPR-Cas system is new gene targeting modification technology developed from bacterial acquired immunity. In this study, MSTN gene was knockouted by CRISPR Cas9 system in goat fetal fibroblast cells and muscle satellite cells. Then gene linked to muscle development tested by real time quantitative PCR and Western blot after the MSTN gene knock out. It is offers a new method for sheep breeding.1. Design and construction of gRNA expression vector and transfection efficiency analysis.In this study, four pairs of gRNA with 20nt long were designed trough the CRISPR Design (http://crispr.Mit.edu/) software of the Massachusetts Institute of technology. gRNA-T2 serve as a template for PCR to obtain complete gRNA.In order to select the best adapted to the goat cell transfection conditions, red fluorescent protein expression vector pCMV-DsRed were transfected into cashmere goat fetal fibroblast cells by using electroporation to screent the optimal transfection conditions. It can be observed a large number of positive cells expressed the red fluorescence and the cells grow well under inverted fluorescence microscope after transfection 48h.The results were analyzed by flow cytometry showed that the optimal transfection conditions:for every 1 × 106 fetal cashmere goats fibroblast cells were added pCMV-DsRed plasmid 10μg (1μg/μL), Opti-MEM 90μL, voltage 225V, pulse time 2.5ms.hCas9 vector and gRNA were transfected together into fetal Arbas cashmere goat fibroblast cells Under the optimal transfection conditions. Design Cross-targeting sites PCR primers for PCR amplification, MSTN gene amplificated directly from genome DNA of goat fibroblast cells and three gRNA with higher efficiency were determined. 2. Preparation and analysis of goat fetal fibroblasts and muscle satellite cells with MSTN gene knock-out by CRISPR-Cas9s.Using the most suitable efficiency electroporation transfection conditions, hCas9 vector and gRNA were imported into fetal cashmere goat fibroblast cells and muscle satellite cells. Single cells were randomly picked after transfection and cultivated in 96-well plates to set up monoclonal cell lines. PCR amplifying and sequencing for every genome of monoclonal cell line were applied to select monoclonal cell lines with mutated target sites.In this study,62 monoclonal fetal fibroblasts cell lines were obtained,including 10 monoallelic knockout cell lines and 10 biallelic knockout cell lines,with a total knockout efficiency of 32.25%. Six muscle satellite monoclonal cell lines were obtained, including 5 biallelic knockout positive monoclonal fetal fibroblasts cell lines, with 83.33% efficiency of knockout.On consideration of the above study, this paper chooses the fetal fibroblasts cell of NO.C205, one of the biallelic knockout cell lines, as sample to analyze gene expression. The muscle growth related gene expression level for testing by qPCR in MSTN gene knockout cells. The MSTN gene mRNA expression level decreased 73.79% in this cells, and expression of MSTN protein in MSTN knockout cells is less than wild-type cells. With the reduction of the MSTN expression, the MyoG、Myf5 and Myf6 gene transcription level were increased to different degree. So MSTN may play a negative regulatory role of these three genes.