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Fig. 3 | Genome Biology

Fig. 3

From: Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases

Fig. 3

Comparisons of the specificities of WT SpCas9 and eSpCas9(1.0), eSpCas9(1.1), and SpCas9-HF1 with tRNA–sgRNA constructs. Off-target effects of WT SpCas9 and variants eSpCas9(1.0), eSpCas9(1.1), and SpCas9-HF1 with guide sequences containing pairs of mismatches at successive positions. The WT guide sequence is highlighted in bold with the PAM highlighted in blue. The WT guide sequence was systematically mutated by introducing pairs of mismatches at successive positions. A total of 20 sgRNAs (one WT guide sequence and 19 mutated guide sequences) were produced using U3:tRNA–sgRNA-N20 constructs. Each sgRNA was tested in combination with the four SpCas9 nucleases, and the percentage of indels was used to measure editing activity. The mismatch sites are highlighted in red

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