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

Figure 5. The C-terminal binding domain is sufficient for Cas12a inhibition.

(A) Atomistic models for the AcrVA4, AcrVA4 Δ1–134, LbCas12a-crRNA, LbCas12a-crRNA-AcrVA4 Δ1–134 and LbCas12a-crRNA-AcrVA4 that were used to match experimental small-angle X-ray scattering (SAXS) data shown in panel B and panel C. (B) Experimental data for AcrVA4, AcrVA4 Δ1–134 (red and gray) and theoretical (red line) SAXS profiles for the solution state models shown in the panel A. SAXS fits are shown together with the fit-residuals. (C) Experimental data for LbCas12a-crRNA, LbCas12a-crRNA-AcrVA4 Δ1–134 and LbCas12a-crRNA-AcrVA4 (teal, gray and red) and theoretical (red line) SAXS profiles for the solution state models shown in the panel A. SAXS fits are shown together with the fit-residuals. (B/C-insets) Normalized P(r) determined from the experimental SAXS curves. The area of each P(r) is normalized relative to the SAXS calculated molecular weights (Table 2). (D) LbCas12a dsDNA cis-cleavage over time measured under single-turnover conditions in the presence or absence of AcrVA4 or AcrVA4 Δ1–134 (mean ∓ s.d., n = 3 independent measurements). Two-phase exponential decay experimental fits are shown as solid lines. (E) Schematic representation of phage lambda plaque assay in E. coli. All strains harbor both a CRISPR-Cas plasmid (purple) and anti-CRISPR plasmid (red). Cas12a confers immunity to phage lambda, while anti-CRISPR inhibition of Cas12a restores plaquing. (F) Phage plaque assay to compare inhibition of LbCas12a by wild-type AcrVA4 and mutants relative to positive (AcrVA1: Acr +) and negative (AcrIIA4: Acr -) control anti-CRISPRs in E. coli. Ten-fold serial dilutions of heat-inducible phage lambda spotted on lawns of E. coli strains expressing the specified anti-CRISPR protein and a non-targeting guide (-) or lambda-targeting guide (+). Images shown are representative of the effect seen in replicates (n = 3 independent measurements).

Figure 5.

Figure 5—figure supplement 1. Small-angle X-ray scattering (SAXS) and multi-angle light scattering (MALS) data.

Figure 5—figure supplement 1.

(A) Top, SEC-SAXS profiles for full-length AcrVA4 (red) and the truncated AcrVA4-Δ1–134 (gray). I(0) (lines) and Rg (symbols, Å) are shown for each collected frame across the relevant SEC peak. Bottom, SEC-MALS profiles for full-length AcrVA4 (red) and the truncated AcrVA4-Δ1–134 (gray). Intensity (AU) and calculated Molar Mass (kDa) are shown for each collected frame across the relevant SEC peak. (B) Top, SEC-SAXS profiles for LbCas12a-crRNA in the presence of full-length AcrVA4 (red). I(0) (lines) and Rg (symbols, Å) are shown for each collected frame across the relevant SEC peak. Bottom, SEC-MALS profiles for LbCas12a-crRNA in the presence of full-length AcrVA4 (red). Intensity (AU) and calculated Molar Mass (kDa) are shown for each collected frame across the relevant SEC peak. (C) Normalized Kratky plots for full-length AcrVA4 (red), truncated AcrVA4 Δ1–134 (gray) adjacent to (D) normalized Kratky plots for LbCas12a-crRNA (teal), LbCas12a-crRNA in the presence of full-length AcrVA4 (red), and LbCas12a-crRNA in the presence of truncated AcrVA4 Δ1–134 (gray).
Figure 5—figure supplement 2. Phage lambda plaque assays.

Figure 5—figure supplement 2.

Comparing inhibition of LbCas12a, Lb#Cas12a, AsCas12a, and chimeric As*Cas12a by wild-type AcrVA4 relative to positive (AcrVA1) and negative (AcrIIA4) control anti-CRISPRs in E. coli. Inhibition of Cas12-mediated phage immunity by AcrVA4 mutants was tested for LbCas12a and was not determined (n.d) for Lb#Cas12a, AsCas12a, and As*Cas12a. Ten-fold serial dilutions of heat-inducible phage lambda spotted on lawns of E. coli strains expressing the specified anti-CRISPR protein and a non-targeting guide (-) or lambda-targeting guide (+). Biological triplicates are shown.