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. 2019 Aug 16;2(4):209–222. doi: 10.1089/crispr.2019.0013

FIG. 4.

FIG. 4.

iCas9 targets plasmid-to-plasmid recombination in human cells. (A) A dual-plasmid reporter for detection of intermolecular recombination. A promoterless GFP donor vector contains an iCas9 recognition site. A separate mCherry acceptor vector contains an EF1α-HTLV promoter with iCas9 target site and mCherry downstream. Recombination results in placement of GFP downstream of the promoter and mCherry-GFP dual-positive cells. (B) HEK293Ts were cotransfected with dual-reporter plasmids, iCas9 and sgRNAs. Scale bar = 200 μm. (C) Flow cytometry scatter plots of plasmid-to-plasmid recombination experiments. Untransfected HEK293Ts (gray, lower left, LL) were used to define gates for GFP+ and mCherry+ (dashed lines). HEK293Ts were cotransfected with reporter vectors, iCas9, and sg(NT) (red) or sg(G:H) (blue). Targeting resulted in GFP-mCherry dual-positive cells (upper right, UR). (D) Fold-increase of GFP-mCherry dual-positive cells for iCas9 transfections. Targeting of GFP-donor and mCherry-acceptor with sg(G:H) results in a 10.6 ± 0.5 fold-increase of dual-positive cells, results of recombination, at the target site compared to a control sgRNA sg(NT). ***p < 0.001, as determined by one-tailed Student's t-test.