Fig. 3: Computational analysis and functional validation reveal nucleotide positions within the spacer as key determinants of silencing efficiency.
a, Bioinformatics pipeline exploring the parameters affecting PspCas13b silencing. PFS positions are indicated. A total of 201 crRNAs are ranked by silencing efficiency. Highly potent (>90% efficiency, yellow box) and ineffective (<50% efficiency, blue box) crRNAs were analyzed for PFS and spacer nucleotide positions. b, Position weight matrices (PWMs) depicting the positional nucleotide probabilities of upstream or downstream PFSs in the potent (left) or ineffective (right) crRNAs. c,d, Delta nucleotide probabilities of the potent (c) and ineffective (d) spacer sequences that compare filtered spacer nucleotide positions to the baseline nucleotide distribution. The red color in the spacer sequence highlights the position of key residues. e,h, Prospective design and validation of predicted potent crRNAs harboring a G-G motif at the 5′ end of spacers targeting eGFP (e) and TagBFP (h) (n = 4). f,i, Predicted ineffective crRNAs lacking a 5′ G-G motif and harboring C bases in the central region of spacers targeting eGFP (f; n = 4) and TagBFP (i; n = 3). The red color in the spacer sequence indicates nucleotide insertion or substitution. Data points are the averaged mean fluorescence from four representative fields of view per condition. The data are represented in AU. Errors are the s.e.m. and P values of a one-way ANOVA are indicated (95% confidence interval). g,j, The average silencing efficiency of predicted potent and ineffective crRNAs targeting eGFP (g) and TagBFP (j) transcripts (potent crRNAs, n = 3; inefficient crRNAs, n = 4). Data are the normalized means and errors are the s.e.m. Results were analyzed by an unpaired two-tailed Student’s t-test (95% confidence interval). k, RfxCas13d silencing assay to target mCherry transcript in HEK 293T cells using the top ten potent crRNAs predicted by the RfxCas13d guide prediction platform (Wessels et al.32). NLS, nuclear localization sequence. l, Left, the silencing efficiency of the top ten RfxCas13d crRNAs. Data points represent the averaged mean fluorescence from four representative fields of view per condition (n = 3) in AU. Errors are the s.e.m. and one-way ANOVA P values are indicated (95% confidence interval). Right, the average silencing efficiency of predicted crRNAs. Data points represent the normalized means and errors are the s.e.m. (95% confidence interval) (n = 3).
