Fig. 3: Achieving high specificity by iterative crRNA design.

a Trans-cleavage activity of four DNA base types at position 14 of the non-template strand of three targets was tested against that of various crRNAs with different spacer lengths. b Trans-cleavage activity of four DNA base types and four RNA base types paired at R-loop position 14 for three targets. c The mismatch tolerance of crRNA with a 17 bp spacer was evaluated for different distances from the position 14 mismatched activator; in the case of the three targets, NTS was mutated to C or A, forming a rU-dG or rG-dT wobble pairing at position 14 in the spacer-TS DNA heteroduplex. Heatmap indicating the mean fold change in fluorescence value within 1 h for the activators containing the rU-dG or rG-dT wobble pair at position 14 in the 17 bp spacer-TS DNA heteroduplex, normalized to that of the corresponding distance mismatch activators. The values in the heatmap represent the multiples of specificity. d Schematic workflow of the iterative design of the crRNA spacer sequence based on the NTS sequence of the SNV target.