Evaluation of Homology-Independent CRISPR-Cas9 Off-Target Assessment Methods

FIG. 1.. (A) Overlap among three replicates for sites nominated by GUIDE-seq, CIRCLE-seq, and SITE-seq across all eight gRNAs. (B) Correlation of read counts for nominated sites between two replicates for GUIDE-seq (R2 = 0.54–0.67), CIRCLE-seq (R2 = 0.80–0.83), and SITE-seq (R2 = 0.90–0.91) read counts. The plots present data for replicates 1 and 2, and the R2 range is calculated from all three comparisons (1 vs. 2, 2 vs. 3, 1 vs. 3). (C) Distributions of read counts for nominated sites obtained by for GUIDE-seq, CIRCLE-seq, and SITE-seq grouped by the number of mismatches and gaps in the site with respect to gRNA sequence. gRNA, guide RNA.

FIG. 2.. (A) Overlap among three genome-wide assays for sites nominated across eight gRNAs. (B) Edit distances of nominated sites across eight gRNAs shared among and unique to different homology-independent nomination methods. The x-axis represents overlap with the three genome-wide assays. The y-axis shows the sum of mismatches and gaps for a site when aligned to the gRNA sequence. All three distributions were significantly different from each other (Wilcoxon p-value <2e-16).

FIG. 3.. Overlap between the number of sites nominated by the three genome-wide assays and sites sequence-confirmed as edited in HEK293T/Cas9 cells following hybrid capture sequencing in the 5 MM 0 gap, 3 MM 1 gap homology space. Sites sequenced with an average depth of more than 500 reads and with <30% indel frequency in the untreated samples are included. MM, mismatch; indel: insertions and deletions.

FIG. 4.. Indel frequencies (%) differences between treated and control samples from three replicates of hybrid capture followed by sequencing in HEK293T/Cas9 cells are shown for all 51 edited sites, ranked by average indel frequency difference. Each color represents one replicate. Vertical bars represent standard deviation in indel frequency difference across replicates. Panels depict overlap with genome-wide assays. The first panel shows one site not nominated by any genome-wide assay. The second panel shows one site nominated by two genome-wide assays. The third panel shows 49 sites nominated by all three genome-wide assays.

FIG. 5.. Correlation of read counts for nominated sites obtained in (A) GUIDE-seq, (B) CIRCLE-seq, and (C) SITE-seq with an average indel frequency (%) difference between treated and control samples from three replicates of hybrid capture followed by sequencing in HEK293T/Cas9 cells. Color images are available online.