Prediction-based highly sensitive CRISPR off-target validation using target-specific DNA enrichment

CRISPR effectors, which comprise a CRISPR-Cas protein and a guide (g)RNA derived from the bacterial immune system, are widely used to induce double-strand breaks in target DNA and activate the in-vivo DNA repair system for target-specific genome editing. When the gRNA recognizes genomic loci with sequences that are similar to the target, deleterious and often carcinogenic mutations can occur. Off-target mutations with a frequency below 0.5% remain mostly undetected by current genome-wide off-target detection techniques. In this study, we developed a method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification. We used various genome editors, including CRISPR-Cpf1, Cas9, and an adenine base editor, to induce intracellular genome mutations. The CRISPR amplification method detected off-target mutations at a significantly higher rate (1.6∼984 fold increase) than did an existing targeted amplicon sequencing method. In the near future, CRISPR amplification in combination with genome-wide off-target detection methods will allow to detect genome editor-induced off-target mutations with high sensitivity and in a non-biased manner.