The pSpCas9(BB)-2A-GFP (PX458) plasmid contained the human codon optimized SpCas9 gene with 2A-EGFP. pSpCas9(BB)-2A-GFP (PX458) was a gift from F. Zhang (Addgene plasmid; catalog no. 48138) (21 (link)). Cloning of sgRNA was done using Bbs I sites. The sgRNAs in this study, listed in table S1, were selected using prediction of crispr.mit.edu . sgRNA sequences were cloned into PX458 and then tested in tissue culture using human embryonic kidney (HEK) 293 cells and 10T½ cells, as previously described (37 (link)).
The AAV TRISPR-sgRNAs-CK8e-GFP plasmid contained three sgRNAs driven by the U6, H1, or 7SK promoter and green florescent protein (GFP) driven by the CK8e regulatory cassette. The TRISPR backbone cloning system relies on two consecutive steps of the Golden Gate Assembly (New England Biolabs). Details of the assembly were previously described (11 (link)).
The AAV TRISPR-sgRNAs-CK8e-GFP plasmid contained three sgRNAs driven by the U6, H1, or 7SK promoter and green florescent protein (GFP) driven by the CK8e regulatory cassette. The TRISPR backbone cloning system relies on two consecutive steps of the Golden Gate Assembly (New England Biolabs). Details of the assembly were previously described (11 (link)).
