Figure 2.

Introduction of NES Peptides to Lower Background Activity of HIT-Cas9

(A) Schematics (not drawn to scale) of various fusion constructs of NES-tagged Cas9-ER^T2. (B) NHEJ-induced CD201 knockout was examined using flow cytometry. ISO, 293T cells incubated with antibody isotype control; CD201, 293T cells incubated with anti-CD201 antibody conjugated with PE-Vio770 fluorophore; ctl sgRNA, cells transfected with an unrelated sgRNA and incubated with the CD201 antibody; PC, cells transfected with Cas9-NLS and CD201 sgRNA and incubated with the CD201 antibody. (C) Cartoon illuminating the working mechanism of a fluorescence conversion reporter (FCR) assay, in which blue fluorescence protein (BFP) is converted to GFP upon HDR-mediated substitution of a key amino acid. (D) HDR efficiency was measured by flow cytometry in the FCR assay. Representative plots (top) and quantifications (right bottom) are shown. NC, cells transfected with an unrelated sgRNA; PC, cells transfected with Cas9-NLS and BFP sgRNA; ctl sgRNA, unrelated sgRNA. (E) Cartoon illuminating the working mechanism of the optimized drug-inducible HIT-Cas9 genome-editing system. Data showed mean ± SD. n = 3 biological replicates. ns, non-significant; **p < 0.01; ****p < 0.0001; Student’s t test.