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. Author manuscript; available in PMC: 2019 Oct 1.
Published in final edited form as: Nat Struct Mol Biol. 2019 Apr 1;26(4):315–321. doi: 10.1038/s41594-019-0208-z

Figure 5 – AcrVA1-triggered endoribonuclease activity is multiple turnover, requires the PID, and competes with AcrVA5.

Figure 5 –

a) Percentage crRNA spacers truncated after 1 hr at different ratios of AcrVA1:Cas12a-crRNA (mean ± sd, n = 3 independent experiments). b) Radiolabeled kinetic crRNA cleavage assays for (left to right) LbCas12a, ∆PID LbCas12a, and ∆REC1/2 LbCas12a in the presence of AcrVA1. Time courses represent 1, 2, 5, 15, and 60 min. Black triangles indicate the full-length and truncated crRNA. The approximate position of the truncated domains is shown with a red cross. c) Quantified time-course of percentage AcrVA1-triggered spacers truncated in the presence AcrVA4 or AcrVA5 (n = 3 independent experiments). Experimental fits are shown as solid lines and the calculated pseudo-first-order rate constants (kobs) (mean ± sd) are 4.8 ± 0.2 min−1, 4.4 ± 0.3 min−1, and 0.5 ± 0.04 min−1 for AcrVA1, AcrVA1 + AcrVA4, and AcrVA1 + AcrVA5, respectively. Source data for panels a and c are available with the paper online. The uncropped gel images are available in Supplementary Data Set 1.