Supplementary Figure 2: Evaluation of R661 residue to improve on-target activity of the VNEL SpCas9 variant
From: A highly specific SpCas9 variant is identified by in vivo screening in yeast
(a) SpCas9 crystal structure (PDB ID: 4OO8) showing arginine 661 and its predicted interactions with the target DNA:guide RNA heteroduplex. While the R661L substitution likely introduces clash interactions (indicated by red disks) with the sgRNA backbone in all its rotamers (a representative rotamer is shown), the R661Q and R661S mutations are predicted to preserve one and two polar contacts with the guide, respectively, and simultaneously disrupt bonding interaction with the target DNA backbone. (b) 293multiEGFP were transfected with wild-type SpCas9, evoCas9 or the VNEL variant together with sgRNAs targeting different regions of the EGFP coding sequence. The loss of EGFP fluorescence (reported as mean percentage) was measured by FACS analysis 7 days post-transfection. (c) Editing of five genomic loci was evaluated in 293T cells after transfection with wild-type SpCas9, the VNEQ (evoCas9) variant or the VNEL variant together with the corresponding sgRNA. Indel formation (reported as mean percentage) was measured using the TIDE tool 7 days post-transfection. In panels (b,c) individual biologically independent samples are represented as overlaid circles.
