Fig. 2: Detection of off-target mutations induced by CRISPR–Cas12a.
From: Prediction-based highly sensitive CRISPR off-target validation using target-specific DNA enrichment
a Detection of off-target mutations for the target sequence (RPL32P3 site) generated by the CRISPR–Cas12a effector in U2OS cells. PCR amplicons were generated for 10 off-target sequences3 similar to the target sequence and the indel frequency (%) was determined by NGS after sequential amplifications with CRISPR–Cas12a. Each round of CRISPR amplifications are marked by different colors (light blue: no amplification, blue: 1st CRISPR amplification, green: 2nd CRISPR amplification, dark green: 3rd CRISPR amplification). NC indicates a negative control for no Cas12a delivery into the cells. 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. The dashed line indicates the NGS detection limit (=0.5%). 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). b Fold increase after CRISPR amplification for each target and off-target sequence. Primary, secondary, and tertiary CRISPR amplification (N = 2) results are shown in gray, dark gray, and black, respectively. Source data is in the Source Data file.
