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

Seung-Hun Kang,Hanseop Kim,Yeung Bae Jin,Bong-Hyun Jun,Junho K Hur,Seung Hwan Lee,Wi-Jae Lee,Young-Ho Park,Sun-Uk Kim,Yeounsun Oh,Ju-Hyun An,Jong-Hee Lee,Young-Hyun Kim

doi:10.1038/s41467-020-17418-8

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.

Highlights

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
The existing next-generation sequencing (NGS) methods have limited sensitivity in detecting genome-wide off-target mutations induced by CRISPR effectors in vivo
To verify that mutated DNA was relatively amplified over non-mutated DNA by this method, we edited HEK293FT cells using the CRISPR–Cas12a system to induce indels in target sequences and applied the CRISPR amplification method to genomic DNA samples extracted from the cells (Fig. 1b–d)

Summary

Introduction

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. Various methods, mostly based on next-generation sequencing (NGS), have been developed to detect the off-target mutations in vivo[5,13,14,15] These methods provide genome-wide detection of off-target mutations in an unbiased fashion both, inside and outside the cell. We develop a method for detecting very small amounts of off-target mutations (below the detection limit of conventional amplicon sequencing) derived from genetic modification of specific sequences using CRISPRamplification technology in vivo with high accuracy

Methods

Results

Conclusion