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

Nature Communications
Seung-Hun KangSeung Hwan Lee

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.

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Citations

Apr 4, 2021·Cancers·Laia Castells-RocaJordi Surrallés
Nov 7, 2020·Cell Stem Cell·Delilah HendriksBenedetta Artegiani
Jun 19, 2021·Molecular Biology Reports·Kirti PrasadKumarasamypet M Mohankumar
Aug 28, 2021·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Hong Jo LeeJae Yong Han
Sep 30, 2021·Molecular Biotechnology·Gokul Kesavan

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Methods Mentioned

BETA
amplicon
genetic modification
PCR
genotyping
GUIDE-seq
amplicon sequencing
electrophoresis
transfection

Software Mentioned

GUIDE
ImageJ
Cas
Graphpad prism
Fastq
OFFinder

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