Figure 6.

The Cutting Efficiency and Specificity of PS822 in Human Hepatocytes

(A) Plasmids encoding SpCas9 and three candidate gRNAs were transfected into human hepatocytes (Huh7 cell line), and the cleavage activity was measured through sequencing the target locus. Only gRNA3 showed significant cleavage, and it was selected as the candidate for further analyses. (B) The plasmid encoding SpCas9 and the donor plasmid encoding IDUA cDNA and gRNA3 were cotransfected into human hepatocytes (HepG2) cells. After 48 h, genomic DNA was extracted from collected cells, and nested PCR was performed. Cells without transfection and cells transfected with the donor plasmid only were used as controls. The gel image of the second round PCR is shown. Only cells transfected with both Cas9 and donor plasmids had the band at the expected size of 1,031 bp. (C) Cell pellets and supernatants were processed for IDUA enzyme assays. IDUA enzyme activities in cells transfected with Cas9 and donor plasmids had significantly higher IDUA enzyme activities in cell pellets and supernatants compared with untransfected cells and cells transfected with the donor plasmid only. (D) SpCas9 and gRNA3 ribonucleoprotein were cotransfected with double-strand oligonucleotide tag (dsTag) into Huh7 cells. Genomic DNA was extracted and used for library preparation. Deep sequencing was performed to search for the dsTag. (E) Only on-target cleavage was identified through GUIDE-seq.