Extended Data Fig. 2: Evaluation of off-target effects of CRISPR–Cas3 using capture sequencing.
From: CRISPR–Cas3-based editing for targeted deletions in a mouse model of transthyretin amyloidosis
(a) Distribution of deletion start sites in Hepa1-6 cells transfected with the Cas3-Cascade expression plasmid (pCas3-crRNA#2), as determined by capture sequencing. (b) Similar analysis of deletion start sites in the liver of ICR mice injected with DiT-Cas3-LNP (6 mg/kg). In both panels, the horizontal axis represents deletion size (in base pairs), and the vertical axis indicates the genomic position of the deletion start site, measured relative to the crRNA binding site (position = 0). (c) Analysis of split reads at potential off-target (POT) sites in Cas3-treated cells. To evaluate off-target activity of Cas3, capture sequencing was performed for 5 kb regions flanking in silico–predicted POT sites (see Fig. 2a and Supplementary Table 7). Split reads detected at POT93 and POT295 were aligned to the mouse genome (mm10) and visualized using Integrative Genomics Viewer (IGV). RepeatMasker tracks revealed that these regions are located near repetitive elements, which may cause misalignment artifacts. Notably, the crRNA spacer sequence showed low homology to these regions, and deletion patterns were not reproducible across replicates. These observations suggest that the weak signals at POT93 and POT295 are likely false positives, rather than genuine Cas3-induced events.
