Pcmv abe xcas9 3.7

The pCMV-ABE7.10, pCMV-ABE-xCas9(3.7) and pCMV-ABEmax were obtained from Addgene. The NG and other mutations in the PAM domain were introduced by fusion PCR of pCMV-ABEmax and subcloned into pCMV-ABEmax. The A56G and V82G mutations were introduced into TadA* domain by fusion PCR. The CfaN minigene was synthesized by IDTdna and fused at the amino acid 573 of SpCas9-max through PCR amplification. The TadA-TadA*-SpCas9max(2-573)-CfaN fragment was PCR amplified and subcloned into pAAV under the control of meCMV promoter to generate pAAV-ABEmaxN-temp. The hU6 promoter with mdx^4cv-targeting gRNA was PCR amplified and cloned into pAAV-ABEmaxN-temp to make pAAV-ABEmaxN. The CfaC fused with SpCas9max(574-end) was generated by PCR and cloned into pAAV-ABEmaxN-temp to make pAAV-ABEmaxC. Similarly, pAAV-ABEmaxN2 and pAAV-ABEmaxC2NG with the Gp41-1 intein, and pAAV-ABEmaxN3 and pAAV-ABEmaxC3NG with the Npu intein were constructed. The mdx^4cv gRNA and other gRNA oligos (listed in Supplementary Table S5) were annealed and ligated into pLenti-ogRNA. The mdx^4cv reporter oligos were annealed and ligated into pLKO-puro-2A-EGFP to form the mdx^4cv reporter plasmid as previously described^{39 (link)}. All plasmids used in this study are listed in Supplementary Table S6.