Figure 7
Identification of on- and off-target indel mutations from human 293T and iPSCs using PAGE-based genotyping protocol.
(A) On-target screening in 293T cells using agarose gel electrophoresis. 293T cells were transfected using Cas9-GFP plasmids with either vehicle (no sgRNA), sgRNA-con (control sgRNA targeting a third locus (data not shown) or sgRNA-ATXN1 (sgRNA targeting the ATXN1 locus). Two days post transfections, cells expressing eGFP were sorted by flow cytometry prior to DNA extraction. (B) On-target screening in 293T cells using the PAGE-based approach. PAGE-based genotyping protocol detected heteroduplex DNA formation in cells transfected with sgRNA-ATXN1, but not in controls. (C) On-target screening in iPSCs using agarose gel electrophoresis. Human iPSCs were electroporated with either vehicle (no sgRNA) or sgRNA-TBP (sgRNA targeting the TBP locus). (D) On-target screening in human induced pluripotent stem cells using PAGE-based approach. (E) Off-target screening in 293T cells using agarose gel electrophoresis. 293T cells were transfected using Cas9-GFP plasmids with either vehicle (no sgRNA), sgRNA-con (control sgRNA targeting a third locus or sgRNA-ATXN2 (sgRNA targeting the ATXN2 locus) for screening off-target loci. Three off-target loci including ZDHHC8, SPOCK2 and WNT6 were identified using CRISPR Design Tool (http://crispr.mit.edu). (F) Off-target screening in 293T cells using PAGE-based analysis. Note that off-target effect was only detected in the WNT6 locus, not in controls or other loci tested. Heteroduplex DNA bands were marked by asterisks in (B), (D) and (F).
