Fig. 3: Detection of off-target mutations induced by CRISPR–Cas9.
From: Prediction-based highly sensitive CRISPR off-target validation using target-specific DNA enrichment
a Left: detection of off-target mutations for the target sequence (FAT3 site1) generated by the CRISPR–Cas9 effector in HEK293FT cells. PCR amplicons were generated for on-target and three off-target sequences predicted in silico and the indel frequency (%) was analyzed by NGS after sequential CRISPR amplifications. The Y axis represents the amplified target and off-target sequences, and the X axis represents the indel frequency (%) in the analyzed amplicon on a log scale. NC indicates a negative control for no CRISPR–Cas9 delivery into the cells. Each amplification stage for mutant DNA enrichment is shown in gray (NC), light blue (no amplification), cyon (1st CRISPR amplification), green (2nd CRISPR amplification), and dark green (3rd CRISPR amplification). The dashed line indicates the NGS detection limit (=0.5%). All experiments were conducted at least two times. Data are shown as mean from two (N = 2) independent experiments. P-values are calculated using a one-way ANOVA, Tukey’s test (ns: not significant, *P = 0.0332, **P = 0.0021, ***P = 0.0002, ****P = 0.0001). Right: schematics of the target-specific editing with Cas9 and mutant DNA enrichment with Cas12a, respectively. b Fold increases in FAT3 target and off-target mutant DNA after CRISPR amplification (N = 2). c Left: detection of off-target mutations for the target sequence (HEK293 site4) generated by the CRISPR–Cas9 effector in HEK293FT cells. PCR amplicons were generated for on-target and six off-target sequences according to the previous study5 and the indel frequency (%) was analyzed by NGS after sequential CRISPR amplifications. Each amplification stage for mutant DNA enrichment is shown in gray (NC), light pink (no amplification), dark pink (1st CRISPR amplification), red (2nd CRISPR amplification), and dark red (3rd CRISPR amplification). All experiments were conducted at least two times. Data are shown as mean from two (N = 2) independent experiments. P-values are calculated as in a. Right: schematics of the target-specific editing with Cas9 and mutant DNA enrichment with Cas9, respectively. d Fold increases in target (HEK293 site4) and off-target mutant DNA after CRISPR amplification (N = 2). In b and d, primary, secondary, and tertiary CRISPR amplification fold increase are shown in light gray, dark gray, and black. Source data is in the Source Data file.
