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

Seung-Hun Kang, Wi-jae Lee, Ju-Hyun An, Jong-Hee Lee, Young-Hyun Kim, Hanseop Kim, Yeounsun Oh, Young-Ho Park, Yeung Bae Jin, Bong-Hyun Jun, Junho K. Hur (), Sun-Uk Kim () and Seung Hwan Lee ()
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Seung-Hun Kang: Kyung Hee University
Wi-jae Lee: Korea Research Institute of Bioscience and Biotechnology (KRIBB)
Ju-Hyun An: Korea Research Institute of Bioscience and Biotechnology (KRIBB)
Jong-Hee Lee: Korea Research Institute of Bioscience and Biotechnology (KRIBB)
Young-Hyun Kim: Korea University of Science and Technology (UST)
Hanseop Kim: Korea Research Institute of Bioscience and Biotechnology (KRIBB)
Yeounsun Oh: Korea Research Institute of Bioscience and Biotechnology (KRIBB)
Young-Ho Park: Korea Research Institute of Bioscience and Biotechnology (KRIBB)
Yeung Bae Jin: Korea Research Institute of Bioscience and Biotechnology (KRIBB)
Bong-Hyun Jun: Konkuk University
Junho K. Hur: Kyung Hee University
Sun-Uk Kim: Korea Research Institute of Bioscience and Biotechnology (KRIBB)
Seung Hwan Lee: Korea Research Institute of Bioscience and Biotechnology (KRIBB)

Nature Communications, 2020, vol. 11, issue 1, 1-11

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

Date: 2020
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DOI: 10.1038/s41467-020-17418-8

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