Figure 6.

Pooling of crRNA enables sensitive cas-directed detection of pathogen nucleic acids

(A) Trans-RNase activity of Cas13a bound to individual precursor crRNA, then reacted with target RNA and trans-RNA U₁₀. Filled symbols indicate 13 crRNA selected for pooling. (B) Trans-RNase activity of Cas13a bound to single or 13 pooled crRNA, then reacted with 13 pooled target RNA and U₂₀ (left) or RNaseAlert (right). (Insets) Linear fit to yield k_app (Table S5). (C) Trans-DNase activity of Cas12a bound to crRNA-1, then reacted with IS2404 or genomic DNA and trans-ssDNA C₁₀. Genomic DNA isolated from M. ulcerans (Mu) contains IS2404 protospacers targeted by crRNA-1, whereas genomic DNA isolated from M. tuberculosis (Mt), E. coli (Ec), and humans (Hs) do not. (D) Trans-DNase activity of components tested on C₁₀. Protospacer in target IS2404 complementary to crRNA-1 abuts a PAM, whereas that of crRNA-4 does not. (E) Trans-DNase activity of Cas12a bound to individual crRNA, then reacted with IS2404, which contains protospacers for each crRNA, and C₁₀. Colors indicate 20 crRNA selected for sub-pooling. Activity of Cas12a-crRNA-4 is indicated by black symbol. (F–G) Trans-DNase activity of Cas12a bound to crRNA pools added cumulatively (F) or together (G), then reacted with IS2404 and C₁₀. Linear fits were used to calculate k_app (see Table S5). (H) IS2404 in DNA samples isolated from 38 patient skin swabs quantified by Cas12a trans-activity (y axis) or by qPCR (x axis). Color-coded patient qPCR status was determined from DNA extracted initially at test site. Solid line, detailed in Inset, indicates linear fit to yield k_app (Table S5); gray shading indicates 95% confidence interval. In all panels data represent mean ± SD. See also Figure S6 and Table S5.