Generation of AAVS1-pur-CAG-hM4Di-mCherry Donor Plasmid

Human codon-optimized Streptococcus pyogenes wild-type Cas9 (Cas9-2A-GFP) and sgRNA T2 were obtained from Addgene (plasmid #44719, plasmid#41818)^{53 (link),54 (link)}. To generate AAVS1-pur-CAG-EGFP donor plasmid, we replaced the hrGFP gene in the AAVS1-pur-CAG-hrGFP plasmid (Addgene plasmid #52344)^{55 (link)} with EGFP gene and inserted woodchuck hepatitis post-transcriptional regulatory element (WPRE) and human growth hormone (hGH) Poly A into the 3’ terminal of EGFP gene to obtain AAVS1-pur-CAG-EGFP donor plasmid. We next amplified hM4Di-mCherry cDNA by PCR from AAV-DIO-hM4Di-mCherry plasmid (a gift from Dr. Bryan L. Roth), respectively. hM4Di-mCherry was inserted into the AAVS1-pur-CAG-EGFP donor plasmid to replace EGFP to get the AAVS1-pur-CAG-hM4Di-mCherry donor plasmid.