Targeted editing of intronic-splicing silencer enhancement of SMN2 Exon 7 inclusion by CRISPR/Case 9

Spinal muscular atrophy(SMA)is an autosomal recessive hereditary neuromuscular disease.Exon 7 and 8 of survival of motor neuron 1(SMN1)gene or only exon 7 homology deletion leads to the failure to produce a full-length SMN gene.The copy number of SMN2 gene with high homology of SMN1 affects the degree of disease and was the target gene for targeting therapy,in which splicing silencer in intron 7 was the key to suppress the inclusion of exon 7.In this study,we projected to use CRISPR/Case 9 for the targeted editing of intronic-splicing silencer(ISS)sequence to promote the inclusion of SMN2 exon 7 and increase the production of SMN2 full-length(FL)gene expression.It happens that there was a protospacer adjacent motif(PAM)at one end of the ISS sequence according to the design of sgRNA.The recombinant vector of sgRNA HSMN2 CRISPR/Case 9 was constructed and transfected into HEK293 cells.Sequencing results showed that the ISS sequence could be edited accurately and targeting in the predicted direction,in which deleting small fragments,inserting small amounts and mutation.Quantitative analysis of RT-PCR products by restriction enzyme of DdeI digestion showed that the FL of SMN2 increased by 8%(P<0.05).In the primary cultured chondrocytes of SMA mice,in which sgRNA HSMN2 CRISPR/Case9 recombinant vector transfection could increase the SMN2 FL gene by 23%(P<0.05)and significantly improve SMN protein levels(P<0.05).CRISPR/Case 9 is an effective tool for gene editing and therapy of hereditary diseases,but it is rarely reported in the treatment of SMA diseases.This study shows that CRISPR/Case 9 was first used for the precision target of ISS sequence editing,which can effectively promote the production of SMN2 FL gene expressions,in which there was an important clinical reference value.