Figure 2.

Cas13a cleavage of target RNA and activation of trans-RNase

(A) Cas13a cleavage (yellow triangle) of dual-labeled target. Components tested on Fluorescent Target RNA-1 containing sequence targeted by crRNA-1 but not −2. (B) Target-activated Cas13a cleavage of dual-labeled trans-RNA U₁₀. (C–E) Single-turnover target cleavage using 1.0 nM Fluorescent Target RNA-1 reacted with varied Cas13a bound to mature (C) or precursor (D) crRNA-1 or varied Fluorescent Target RNA-1 reacted with 5.0 nM Cas13a bound to precursor crRNA-1 (E). Lines show single-exponential fit to yield k_obs. (F–G) Simultaneously recorded target and trans cleavage: RNP reacted with a mixture of Alexa 488 (F₁)-tagged target RNA and Alexa 594 (F₂)-tagged trans-substrate. Time courses were recorded on 5.0 nM Cas13a bound to mature or precursor crRNA-1, then reacted with a mixture of 1.0 nM Alexa 488-labeled Target RNA-1 and varied Alexa 594-labeled trans-RNA U₁₀. Lines show single-exponential fit (where applicable) for target (top) or trans cleavage (bottom) to yield k_obs. In panels (A–C and E) data represent mean ± standard deviation (SD). See also Figures S2 and S3.