Fig. 6: Comprehensive mutagenesis of the PspCas13b spacer–target interaction reveals the interface between mismatch tolerance and loss of activity.
a–g, Comprehensive analysis of spacer–target interaction examining the specificity and mismatch tolerance of PspCas13b at various positions of the crRNA spacer sequence. Perturbation of spacer–target interaction through spacer mutagenesis to introduce consecutive mismatches of 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 nucleotides at the 3′ end (a) and 5′ end (b) of the spacer. Perturbation of spacer–target interaction through spacer mutagenesis to introduce consecutive mismatches of six (c), five (d), four (e) and three (f) nucleotides at various positions of the spacer. Perturbation of spacer–target interaction through spacer mutagenesis to introduce various numbers of nonconsecutive nucleotide mismatches at various positions (g). MSM, mismatch. The nucleotides in red highlight mismatch positions in the spacer sequence. Data points in the graph are averages of the mean fluorescence from four representative fields of view per condition imaged (a,b, n = 5; c–f, n = 4; g, n = 3). The data are represented in AU. Errors are the s.e.m. and P values from a one-way ANOVA are indicated (95% confidence interval).
