Fig. 1

SaCas9 PAM characterization and gRNA spacer length assessment in HEK293, HEK293T, HEK293FT, and HEK293-GFP cells. a A plasmid-based, luciferase knockdown assay in which an invariant target sequence with variable PAMs was placed at the 5′ end of luciferase. Data are shown as means ± standard error of the mean (SEM) (N = 3). b T7E1-measured indel rates resulting from SaCas9 directed to endogenous targets with NNGRRT and NNGRRV PAMs. Data are shown as means ± SEM (N = 2). An unpaired t-test for the two groups yields a P value of <0.0001. c Comparisons of indel rates between SaCas9 and SpCas9 at targets with overlapping (NGGRR(T)) PAMs. Data are shown as means ± SEM (N = 2). N.D. none detected. d Indel rates resulting from SaCas9 directed to endogenous VEGFA (top) and CCR5 (bottom) targets, with gRNAs of varying spacer length. “Sibling” gRNAs target the same precise locus, initiate with a target-matching G, and are marked with same-colored dots. Orange bars represent mean cleavage (±SEM (N ≥ 3)) for gRNAs of that length. N.D. none detected. Middle: Knockdown of green fluorescent protein (GFP) in HEK293-GFP cells as measured by percentage of cell population that is GFP-negative 3.5 days post-transfection