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. 2019 Aug 9;8:e49110. doi: 10.7554/eLife.49110

Figure 2. AcrVA4 recognizes the crRNA 5’-OH and Cas12a crRNA processing nuclease.

(A) AcrVA4 (surface) is shown on the LbCas12a WED domain (cartoon, yellow) and crRNA (blue, cartoon) braced against the bridge-helix (green, cartoon), RuvC (teal, cartoon), REC1 (cartoon, gray), and REC2 domains (cartoon, dark gray). (B) Detailed atomistic view of AcrVA4 (red) recognition of the WED domain pre-crRNA processing nuclease (yellow) and crRNA 5’OH (blue). (C) LbCas12a dsDNA cis-cleavage over time measured under single-turnover conditions in the presence or absence of AcrVA4 containing alanine substitutions (mean ∓ s.d., n = 3 independent measurements). Two-phase exponential decay experimental fits are shown as solid or dashed lines. (D) Detailed atomistic view of the AcrVA4 (red) interface with the LbCas12a RuvC (teal), REC2 (dark gray), and WED domains (yellow). (E) Bar-graph illustrating the apparent rate of LbCas12a-mediated dsDNA cis-cleavage under single-turnover conditions guided by a crRNA bearing a 5’-OH, 5’-PO4 or 5’-dT (−21) in the presence or absence of AcrVA4 (mean ∓ s.d., n = 3 independent measurements).

Figure 2.

Figure 2—figure supplement 1. Extended interaction network between the AcrVA4 CBD and the LbCas12a-crRNA complex WED domain.

Figure 2—figure supplement 1.

(left) LbCas12a (surface representation) bound to crRNA (cartoon, blue/orange) and AcrVA4 (cartoon, red). (right) An extended main chain β-sheet interface between AcrVA4 β3-β4 (sticks, red) and the LbCas12a WED domain (surface and sticks, yellow).
Figure 2—figure supplement 2. Size exclusion chromatography (SEC) traces for AcrVA4 mutants used in this study and the effect of 5'-crRNA chemistry on AcrVA4 binding to LbCas12a.

Figure 2—figure supplement 2.

(A) SEC traces for AcrVA4 mutants purified and used in this study plotted as absorbance (AU) at 260 (dashed) and 280 (solid) nm against elution volume (mL). (B) SEC traces for LbCas12a and/or AcrVA4 (VA4) in the presence of crRNA bearing modification to the 5’ end chemistry. For each plot, the absorbance (AU) at 260 nm (gray) and 280 nm (blue) are shown against elution volume (mL).