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. 2020 Feb 6;7(6):1902312. doi: 10.1002/advs.201902312

Figure 1.

Figure 1

Mechanism of off‐target editing. CRISPR/Cas9 systems accept at least three mismatches in a 20 bp DNA target sequence. Cas9 may introduce unexpected off‐target mutations because sgRNAs recognize DNA sequences with one to a few nucleotide mismatches. a,b) PAM binding and recognition mediates the initial Cas9–DNA interaction, which leads to PAM‐proximal DNA melting. c) gRNA binding on target site initiates DNA unwinding. d) A stable R‐loop was formed between the crRNA and potential target sites. e) The sgRNA sequence recognizes partial mismatches outside the seed sequence instead of on‐target sites. f) Off‐target editing is generated as a result of mismatch recognition by sgRNA. Created with BioRender.com.