Supplementary Figure 4: gRNA binding enhances Cas9 delivery by electroporation.
From: Guide Swap enables genome-scale pooled CRISPR–Cas9 screening in human primary cells
(a) Representative histograms of Cas9 FACS staining. protK = proteinase K. perm = permeabilization. Representative of two independent experiments with similar results. (b-c) CD34 + HSPC were transduced with lenti gRNA CD45_B, then electroporated with 6 µg of Cas9 and 6 µg of RNA as indicated. cr = crRNA. tr = tracrRNA. scram = scrambled sequence RNA. sg = single synthetic guide. The experiment was repeated once with similar results. (b) Fold change in MFI (median fluorescence intensity) of intracellular Cas9 FACS staining, compared to appropriate non-permeabilized control for each condition. n = 3 technical replicates, data are presented as mean. (c) Flow cytometry analysis of CD45 knockout efficiency 4 days post-electroporation. Lenti gRNA-expressing cells were gated using RFP. n = 3 technical replicates, data are presented as mean. (d, e) Agarose gel electrophoresis analysis of indicated dsDNA templates cleaved by indicated Cas9-gRNA RNP complexes. Mtap = Mtap_A. * = uncleaved DNA template. <= excess undigested RNA. dg = split gRNA. IVT = in vitro transcribed gRNA; representative of two independent experiments.
