Packaging Of AAV-SaCas9-sgRNA Adeno-associated Virus And Its Knockout Of MSTN Gene In Balb-c Mice

Genome editing technology(genome editing)is one of the most important methods to study gene function,which is one of the most important genetic manipulation techniques which can modify the DNA sequence at the genomic level.Transfer vectors are needed for gene editing in vivo.Because the transfection efficiency of non-viral vectors is relatively low,it is urgent to find an efficient and safe viral transport vector.AAV vector is regarded as the most potential virus vector.It has many advantages,such as unintegration into host genome,low immunogenicity and no pathogenicity,but its carrying capacity is small(about 4.7 kb).As a simple,flexible and efficient gene editing technique,CRISPR-Cas9 can be used in gene therapy.A large fragment of SpCas9 was discovered at first(about 4.2 kb),and a 3.2 kb SaCas9 was later discovered,making it possible for AAV to mediate the CRISPR-Cas9 system.MSTN is a negative regulatory factor for muscle growth.The loss of function of this gene causes a double muscle phenotype in veterinary animals,thereby increasing the meat production rate.At present,the potential functions of MSTN and its regulation mechanism are still poorly understood.In order to further study the mechanism of MSTN,the following experiments were carried out in this study:1.In this experiment,three sgRNAs targeting the MSTN gene were designed to construct pX601-SaCas9-sgRNA recombinant vector.Using human renal epithelial cell line(HEK293T)and mouse embryonic fibroblast cell line(NI H/3T3),the efficiency of gene editing was detected by T7 El digestion and sequencing,and the knockout efficiency of MSTN protein was identified by Western Blot.2.The pX601-SaCas9-sgRNA was packaged with the virus-assisted plasmid pAAV9-RC,pAAV9-helper,and the recombinant AAV virus was isolated and purified and the virus titer was detected by QPCR.3.The AAV viruses were infected with NIH3T3 cells.After 5 days,the efficiency of gene editing was detected by T7 El digestion and sequencing,and the knockout efficiency of MSTN protein was identified by Western blot.4.The recombinant AAV virus was infected with Balb-c mice.After 6 weeks,the skeletal muscles of the legs were taken.The genomic DNA was extracted and the morphological changes of muscle fibers were observed by paraffin section combined with HE staining.The efficiency of gene editing was detected by T7 El digestion and sequencing.At the same time,the knockout efficiency of MSTN protein was identified by Western blot.The results show that:1.three recombinant vectors pX601-Sacas9-sgRNA1,pX601-Sacas9-sgRNA2 and pX601-Sacas9-sgRNA3 targeting the exon of the MSTN gene are successfully constructed,which proved that they could edit the MSTN gene at the cell level,which resulted in the decrease of the expression of MSTN protein.2.Three recombinant AAV virus AAV-SaCas9-sgRNA1,AAV-SaCas9-sgRNA2,and AAV-SaCas9-sgRNA3 were successfully packaged and the titer was higher than 1010 vg/mL.3.The packaged three recombinant AAV viruses have the ability to edit MSTN gene at the cell level,and can reduce the expression of MSTN protein.4.The recombinant AAV virus AAV-SaCas9-sgRNA 1,AAV-Sacas9-sgRNA2,and AAV-SaCas9-sgRNA3 was successfully infected in mice,and the MSTN gene in the leg muscles of mice was edited to decrease the expression of MSTN protein,which resulted in significant changes in muscle traits.