Fig. 3: Validation of x-gRNAs identified from SECRETS.
From: Selection of extended CRISPR RNAs with enhanced targeting and specificity
a All of the top five x-gRNAs exhibit remarkable on-target activity (comparable or greater than enhanced-specificity Cas9 variant eCas9) and greatly reduced off-target activities across four known off-target sites for the standard x-gRNA during in vitro cleavage assays using purified Cas9 and in vitro-transcribed (x-)gRNAs. n = 3 replicates each, dots are individual data points. Error bars = standard error of the mean. Black arrows highlight off-target activity of Cas9 and eCas9 with the sgRNA that was blocked when using x-gRNAs. b CHANGE-seq5 reveals that x-gRNAs identified through SECRETS exhibit minimal and greatly reduced genome-wide off-target activity, with no novel off-target activity identified relative to the standard gRNA. n = 2 biological replicates each. c x-gRNAs identified through SECRETS exhibit robust genome editing activity following transfection of purified Cas9 ribonucleoprotein (RNP) complexes. sg standard gRNA, dCas9 catalytically inactive Cas9, eCas9 enhanced specificity Cas9, NGS next-generation (illumina) sequencing, T7E1 T7E1 mutation detection assay. n = 3 biological replicates, 2 NGS replicates, and 3 T7E1 replicates. Error bars = standard error of the mean.
