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. 2016 Jul 25;44(19):e149. doi: 10.1093/nar/gkw660

Figure 4.

Figure 4.

Limiting duration of Cas9 induction reduced potential off-target effects. (A and B) HeLa/iCas9-c1 cells were transduced with LentiGuide carrying the indicated sgRNA. Cells were then treated with 0.1 μg/ml DOX for the indicated time, harvested after 96 h and analyzed by western blots. (C–F) HeLa/iCas9-c1 cells were transduced with LentiGuide carrying the indicated sgRNA, treated with 0.1 μg/ml DOX for 12 h and harvested after 48 h for western blot analyses (for iCas9). HeLa cells were transduced with LentiCRISPRv1 carrying the indicated sgRNA and harvested for western blot analyses within 10 days after transduction (Const., for constitutively expressed Cas9). (G and H) HeLa/iCas9-c1 cells were transduced with LentiGuide carrying the indicated sgRNA (inducible). HeLa cells were transduced with LentiCRISPRv1 carrying constitutively expressed Cas9 and the indicated sgRNA (Const.). iCas9 cells were then treated with 0.1 μg/ml DOX for 12 h and cultured for another 60 h. All cells were harvested for genomic DNA isolation and analyzed by T7 endonuclease assay. Stars (*) indicate undigested DNA. Pound signs (#) indicate digested products. The second digested fragments in Figure 4H were too short to be visible in same gel.