Methods for verifying off-target sites of gene editing of the CRISPR system. (A) Methods of guide RNA accuracy verification by in-silico approach. For the target sequence of a specific guide RNA, database-based similar off-target candidates are previously investigated, and whether off-target mutations are actually occur is confirmed in cell level. (B) Top: Methods for detecting intracellular off-target sites of CRISPR effectors. Using the CRISPR system, DNA double strand break is induced at every on-/off-target sites within the cell, captured in various ways, the total number and patterns are investigated. Bottom: Cell-free off-target detection methods of CRISPR effectors. After extracting genomic DNA from target cells, induce DNA double strand break at each on-/off-target sites using the CRISPR system, capture it in various ways, and investigate the entire number and patterns. Images were created with BioRender.com.
