Supplementary Figure 3: evoCas9 intracellular expression and titration of activity
From: A highly specific SpCas9 variant is identified by in vivo screening in yeast
(a) Representative western blot of lysates from 293T cells transfected with wild-type SpCas9, evoCas9 or the other high-fidelity variants, as indicated, together with the sgGFPon sgRNA. The graph below the blot reports the mean densitometric quantification of SpCas9 expression normalized on tubulin levels. Tubulin was used as a loading control. SpCas9 was detected using an anti-FLAG antibody. (b) Titration curve of evoCas9 activity in 293multiEGFP cells. Different amounts of evoCas9 or wild-type SpCas9 were transfected together with a fixed quantity of a sgRNA targeting the EGFP CDS (sgGFPon). Loss of EGFP fluorescence (reported as mean percentage) was measured by FACS at 7 days post-transfection. (c) Representative western blot of lysates from 293T cells transduced with lentiviral vectors expressing either wild-type SpCas9, evoCas9 or SpCas9-HF1, as indicated, and the sgGFPon sgRNA. The graph below the blot reports the mean densitometric quantification of SpCas9 expression normalized on actin levels. Actin was used as a loading control. SpCas9 was detected using an anti-FLAG antibody. Lysates were collected 20 days post-transduction and selection for stable SpCas9 expression. In all panels n=2 biologically independent samples (reported as overlaid circles).
