Extended Data Fig. 10: Genome-wide specificity analysis of CRISPR–Cas3 and Cas9 editing in human HepG2 cells using short- and long-read whole-genome sequencing.
From: CRISPR–Cas3-based editing for targeted deletions in a mouse model of transthyretin amyloidosis
(a) The number of predicted potential off-target (POT) sites identified using GGGenome for Cas3-crRNA. (b) Summary of short-read whole-genome sequencing (WGS) coverage and mapping statistics for HepG2 cells transfected with pCas3 carrying the UT-05 crRNA, and for untransfected controls. The average sequencing depth was 139.4×, providing high sensitivity for structural variant detection. (c) A total of 3,035 candidate deletion regions with a split-read ratio ≥6.44 (corresponding to estimated editing efficiency ≥4%) were identified. Among these, 15 overlapped with POT sites. (d) Genome-wide distribution of candidate deletion events revealed a single prominent peak corresponding to the on-target site, with no other significant signals across the genome. (e) IGV snapshot of a representative candidate region among the 15 overlapping POT sites. All observed split-read signals were confined within <1 kb and mapped to low-complexity or repetitive genomic regions. No reproducible structural variants were detected across biological replicates. (f) Oxford Nanopore long-read WGS in Cas3-crRNA UT05 and Cas9-gRNA NTLA2001 transfected HepG2 cells. (g) Cas3-mediated deletions were restricted to the on-target locus under the tested conditions, with no detectable off-target large deletions elsewhere in the genome. Off-target mutations identified in Cas9-edited HepG2 cells using whole-genome sequencing (h-i). (h) Summary of predicted potential off-target (POT) sites for Cas9-gRNA NTLA2001 as identified by Cas-OFFinder. (i) Detection of the on-target deletion at the human TTR locus by short-read whole-genome sequencing (WGS). (j) Among the computationally predicted off-target sites, three loci—POT93, POT200 (both intergenic), and POT88 (intron of PLCB4)—showed measurable deletion signals. The table summarizes chromosomal location, strand orientation, locus annotation, and deletion frequency relative to the on-target site. These off-target events occurred at lower frequencies than the on-target editing and were confined to noncoding or intronic regions.
