Extended Data Fig. 8: Comprehensive mutagenesis of PspCas13b and RfxCas13d spacer-target interaction revealed that shorter crRNA spacers tolerated fewer mismatches.
(a) Perturbation of spacer-target interaction through spacer mutagenesis to introduce 3-nucleotide consecutive mismatch at various positions of the RfxCas13d crRNA1 (Fig. 3l) spacer; N = 3. (b) Perturbation of spacer-target interaction through spacer mutagenesis to introduce various numbers of non-consecutive nucleotide mismatches at various positions of the RfxCas13d crRNA1 spacer. The nucleotides in red highlight mismatch positions in the spacer sequence; N = 3. Silencing of mcherry transcripts by PspCas13b with a serial shorten crRNAs at the (c) 5’ end and (d) 3’ end of the spacer (based on PspCas13b crRNA12 in Fig. 1d); N = 4. (e) Perturbation of spacer-target interaction through spacer mutagenesis to introduce 3-nucleotide consecutive mismatch at various positions of the 3’ truncated, 27-nt crRNA spacer (crRNA12 Δ28–30); N = 4. (f) Perturbation of spacer-target interaction through spacer mutagenesis to introduce various numbers of non-consecutive nucleotide mismatches at various positions of the 3’ truncated, 27-nt crRNA spacer; N = 3. Data points in the graph are averages of mean fluorescence from 4 representative fields of view per condition imaged. The data are represented in arbitrary units (A.U.). Errors are SEM and p-values of one-way Anova test are indicated (95% confidence interval). N is the number of independent biological replicates. Source data are provided as a Source data file. Note: As opposed to PspCas13b, RfxCas13d tolerated consecutive 3-nt mismatches at the 5’end of its spacer but failed to tolerate consecutive 3-nt mismatches at other positions. Single nucleotide mismatches were fully tolerated at positions 6 and 12, but were only partially tolerated at position 16. Two non-consecutive nucleotide mismatches were also partially tolerated (position 8, 16). 3, 4, and 5 non-consecutive nucleotide mismatches led to a complete loss of silencing.
