Fig. 2.

BNA^NC incorporation broadly improves specificity in vitro. Heat maps showing DNA cleavage specificity scores across >10¹² off-target sequences for either unmodified (top) or BNA^NC-modified crRNAs (bottom) targeting a WAS or b EMX1. Specificity scores of 1.0 (dark blue) correspond to 100% enrichment for, while scores of −1.0 (dark red) correspond to 100% enrichment against a specific base-pair at a specific position. Black boxes denote the intended target nucleotides. Bar graph showing the quantitative difference in specificity score at each position in the 20 base-pair target site and 2 base-pair PAM (N of NGG excluded), between the unmodified and BNA^NC-modified crRNA for c WAS or d EMX1 target sequences. A score of zero indicates no change in specificity. Difference in specificity was calculated as specificity score_BNA^NC−specificity score_RNA. Experiments were performed with 200 nM pre-selection library and 1000 nM Cas9 RNP complex