CRISPR-Cas9 Gene Editing Protocols

The pCMVSau plasmid expressing a human codon optimized SaCas9 and a customizable U6-driven gRNA scaffold have been previously described [18 (link)]. Cognate luciferase indicator constructs were generated as previously described [14 (link)]. Maps of these plasmids and all other SaCas9 plasmids are shown in Figure S1 in Additional file 1.
gRNA used in Fig. 1a was generated by cloning annealed oligos containing the target sequence into pCMVSau. gRNAs used for data shown in Figs. 1b–d and 2d were generated by PCR and transfected as amplicons containing U6 promoter, spacer sequence, and TRACR scaffold. gRNAs used for data shown in Figs. 2b, c and 4a, b were generated by ligating either one or two of these into a pUC19 backbone vector via Gibson Assembly (New England Biolabs).
AAV vectors used in Fig. 3a–c were constructed by Gibson Assembly of one or two gRNA cassettes into SaCas9-containing AAV backbone pSS3. Vectors used in Fig. 3d–f were constructed by subcloning gRNA cassette pairs from vectors pAF089, pAF091, pAF092 into pSS60. Inverted terminal repeats (ITRs) were confirmed by XmaI digest of the vectors.