CRISPR-Cas9 Genomic Insertion into the AAV Safe Harbor Site by Electroporation of iPSC v1

This protocol provides methods for mutagenizing human induced pluripotent stem cells (iPSC) by inserting sequences of interest into the AAV Safe Harbor site (AAVSH). We custom-designed a gRNA targeting the AAVSH site within the first intron of the PPP12CR1 gene. Insertion into this site interrupts the gRNA targeting sequence, making it resistant to further cleavage. This protocol describes the electroporation of a cas9 protein complexed with this gRNA, along with a drug-selectable donor plasmid, to provide a functional example for integration of any desired sequences into AAVSH site. We found that electroporation of precomplexed gRNA-Cas9 protein produces efficient editing (60-90%) with acceptable cell viability (greater than 50% survival). As an example of our strategy, we inserted a neomycin resistance gene into the AAVSH site. The homologous recombination donor plasmid, available from AddGene, includes insertion sequences flanked by two homology arms, each with ~800 bp of sequence from the PPP12CR1 intron adjacent to the target sequence. Following the left homology arm are: (1) a splice acceptor, (2) a T2A ribosome skipping sequence, (3) a NeoR/KanR drug-resistance sequence, and (4) a poly(A) signal. Once inserted into the AAVSH site, exon 1 of the PPP12CR1 gene is spliced to the splice acceptor, producing an mRNA that provides G418 resistance. Any other payload could be delivered using a similar strategy.