The RAAV-SaCas9 Gene Editing System Was Used To Knock Out The Myostatin Gene Of Muscle Cells And Evaluate Its Effect On The Improvement Of Muscle Atrophy

Objective: The integrated AAV-Cas9-sg RNA system has advanta ges such as low immunogenicity,wide host range,high safety,stable expression for a long time and fast construction etc.and has been applied in genome editing and gene expression regulation.However,the system's small capacity,low protein expression level,and low gene specific recognition and editing ability limit its application in vivo.At the same time,the CRISPR/Cas9 regulation of TGF-? family signaling pathway affects muscle cell proliferation and differentiation,thereby improving the treatment of cancer,cachexia,muscular dystrophy and other muscular atrophic diseases has broad prospects.Therefore,an ideal AAV-Cas9-sg RNA system is the basis of the application of this technology in gene research and therapy.In this paper,the AAV-Cas9-sg RNA system was optimized to construct the r AAV-Sa Cas9-sg RNA system,and the myostatin gene of muscle cells was knocked out in vivo and in vitro to analyze the morphological changes of muscle cells and the changes of related cytokines,so as to explore the effect of myostatin gene knockout of muscle cells in improving muscle atrophy-related diseases.Methods: 1.We inserted the puromycin resistance gene into the p X601 plasmid,then designed the sg RNA targeting myostatin gene,and constructed the p X601-puro-sg Mstn plasmid with knockout ability.Then,C2C12 cells were transfected,puromycin resistant medium was used for cell screening,and monoclonal cells were cultured by limited concentration dilution method.Finally,monoclonal cell lines with myostatin gene kno ckout were identified.Changes in TGF-? signaling,Akt-m TOR signaling,and key cytokines in MRFs in C2C12 monoclonal cell lines were analyzed by RT-PCR and Western Bloting.The proliferative activity and cell cycle of C2C12 monoclonal cell line were analyz ed by CCK8 and flow cytometry.2.We used the EF1 promoter and t RNA promoter to replace the CMV and U6 promoter in the p X601 plasmid,and constructed a new plasmid R-p X601 containing Sa Cas9.Then,the expression sequence of e-GFP fluorescent protein was in serted into the plasmid R-p X601,and the r AAV-Sa Cas9-sg Mstn was packaged and purified by co-transfection of HEK293 cells with 3 plasmid.The effects of r AAV-Sa Cas9-sg Mstn on myostatin gene and its protein expression were analyzed by gene sequencing and Western Bloting.The effects of r AAV-Sa Cas9-sg Mstn expression were explored in vivo and in vitro at different viral titers and time periods by flow cytometry and fluorescence imaging.Finally,r AAV-Sa Cas9-sg Mstn was applied to the mice model of muscle atrophy and the histological and cytological changes of muscle cells were analyzed.3.Finally,AAV recombinant viruses containing R-p X601 and p X601 were packaged and myostatin gene editing was performed on thigh muscles of the aged mice.Muscle cells and satelli te cells were analyzed by tissue sections and immunofluorescence.The effects of partially knocked out myostatin genes on muscle were studied by analyzing m RNA and protein expressions of related cytokines.Result:1.Plasmid p X601-puro was successfully con structed and connected to sg RNA with myostatin gene knockout.Through T7 digestion identification,sequencing analysis and target efficiency analysis,it is proved that the constructed plasmid has the ability of genome editing.The transfected plasmid p X601-puro-sg Mstn was used to successfully screen C2C12 monoclonal cell line with myostatin knockout.Myostatin expression had an important effect on cell proliferation related cytokines by RT-PCR and Western bloting analysis.Myostatin expression was to promote the proliferation and cell cycle of C2C12 cells by CCK-8 and flow cytometry.2.We demonstrated that the promoter of the r AAV-Sa Cas9 system was capable of stable long-term expression of gene editing elements.And with the increase of r AAVs in vitro and in vivo,the editing efficiency of myostatin and the expression level of e GFP protein increase d.The expression level of e GFP was used to indirectly understand the gene editing efficiency of Cas9.In addition,we demonstrated that partial myostatin knockdown could improve muscle cells in mice model of muscle atrophy.3.We successfully packaged r AAV9 with two systems R-p X601 and p X601,respectively,and demonstrated that they effectively edited myostatin.We observed weight gain,muscle fiber count and cross-sectional area increase by knocking out myostatin in older mice.Myostatin knockdown changed in related cell signaling pathways,increases Myo D Pax 7 and Myo G protein levels,leading to increased satelli te cell activation,and promot ing muscle cell differentiation and proliferation.In addition,myostatin knockdown reduced levels of Mu RF1 and Atrogin1,preventing muscle cell atrophy.Conclusion: We constructed a compact integrated r AAV-Sa Cas9 genome editing system that effectively performs myostatin gene kno ckout in vitro and in vivo.Myostatin gene knockout enhanced the proliferation and differentiation ability of muscle cells and inhibited their apoptosis.Through analysis of related cytokines,it was found that myostatin gene had significant influence on m yostatin regulation growth factor and pro-protein synthesis/degradation factor.This has potential applications in the treatment of chronic wasting diseases such as muscular dystrophy,cancer and age-related muscular dystrophy.