Figure 6. AsCas12a evades AcrVA4 by concealing its pre-crRNA processing nuclease.

(A) The C-terminal binding domain of AcrVA4 (surface) is shown superposed on the AsCas12a structure where it clashes with the WED domain (yellow, cartoon). (B) Schematic representation of the wild-type (Lb and As) and engineered chimeric (Lb* and As*) Cas12a constructs. (C) Bar-graph illustrating the apparent rate of wild-type or chimeric Cas12a-mediated dsDNA cis-cleavage under single-turnover conditions in the presence or absence of AcrVA4 (mean ∓ s.d., n = 3 independent measurements). Single-phase exponential fits to the cleavage kinetics from which the rate was derived can be found in Figure 6—figure supplement 2C. (D) Phage plaque assays to determine susceptibility of chimeric Cas12a to AcrVA4 in E. coli. Ten-fold serial dilutions of heat-inducible phage lambda spotted on lawns of E. coli strains expressing lambda-targeting guide, wild-type or chimeric (*/#) Cas12a, and AcrVA4 or the indicated control anti-CRISPR protein. Images shown are representative of the effect seen in replicates (n = 3 independent measurements). (E) CRISPR-Cas12a inhibition specificity in human cells. Schematic (top panel) showing human cells stably expressing a fluorescence reporter and doxycycline-inducible anti-CRISPR (Acr) constructs. Acr expression blocks genome editing upon transfection of susceptible Cas ribonucleoprotein (RNP) complexes, quantifiable by flow cytometry of mCherry fluorescence. Assessment of editing efficiency in HEK-RC1 reporter cells (bottom panel) expressing GFP or GFP-Acr polycistronic constructs (AcrVA1, AcrVA4, AcrVA5, AcrIIA4, AcrVA4 Δ1–134) and transfected with various Cas9 and Cas12a RNPs targeting the reporter. Note, in contrast to wild-type AsCas12a (As12a), editing by the AsCas12a-chimera (As*12a) was moderately susceptible to AcrVA4 and AcrVA4 Δ1–134 inhibition.

Figure 6—figure supplement 1. Multiple sequence alignment of Cas12a orthologs.

(A) Multiple sequence alignment of LbCas12a (teal), AsCas12a (purple), and other representative orthologs. The sequence the LbCas12a WED domain where AcrVA4 binds is denoted (red) along with the conserved catalytic triad of the pre-crRNA processing nuclease (blue arrows). The site of the additional sequence that occludes AcrVA4 binding is boxed in yellow.

Figure 6—figure supplement 2. Wild-type and chimeric Cas12a pre-crRNA processing and dsDNA cleavage.

(A) Quantified fraction of 5’-radiolabeled pre-crRNA cleaved by wild-type or chimeric (denoted by *) Cas12a over time under single turnover conditions (mean ∓ sd, n = 3, independent experiments). Experimental fits are shown as solid lines and the calculated pseudo-first-order rate constant (k_obs) (mean ∓ sd, n = 3) are plotted in (B). (B) Bar-graph illustrating the apparent rate of wild-type or chimeric (denoted by *) Cas12a-mediated pre-crRNA processing under single-turnover conditions as derived from panel (A). (C) Quantified fraction of 5’-radiolabeled dsRNA cleaved by wild-type or chimeric (denoted by *) Cas12a over time under single turnover conditions in the presence or absence of AcrVA4 (mean ∓ sd, n = 3, independent experiments). Experimental fits are shown as solid lines and the calculated pseudo-first-order rate constant (k_obs) (mean ∓ sd, n = 3) are plotted in Figure 6C.

Figure 6—figure supplement 3. Optimizing induction of Cas12a chimeras in phage lambda plaque assay.

Determining aTc inducer concentration required to observe full As*Cas12a-, Lb*Cas12a-, and Lb^#Cas12a-mediated immunity to phage lambda using E. coli plaque assay in the absence of a type V-A anti-CRISPR. Ten-fold serial dilutions of heat-inducible phage lambda spotted on lawns of E. coli strains expressing the specified anti-CRISPR protein (negative control AcrIIA4 and wild-type AcrVA4) and a non-targeting guide (-) or lambda-targeting guide (+). Inducer concentrations achieving full immunity without growth deficiency are highlighted (red) and indicate optimum conditions shown in Figure 6D and Figure 5—figure supplement 2. Images are representative of biological triplicates.

Figure 6—figure supplement 4. Human CRISPR-Cas and anti-CRISPR (Acr) genome editing assay.

(A) Monoclonal human genome editing reporter cell lines. Flow cytometry analysis of mCherry fluorescence percentage and median fluorescence intensity (MFI) in various monoclonal cell lines derived from HEK293T stably transduced with the lentiviral vector pCF525-EF1a-Hygro-P2A-mCherry and selected on hygromycin B. Clone 1, referred to as HEK-RC1, was selected for further work and used for CRISPR-Cas and Acr assays. (B) Quantification of guide RNA efficiencies for AsCas12a and SpCas9 RNP-mediated genome editing. HEK-RC1 reporter cells were transiently transfected with various AsCas12a or SpCas9 RNP complexes targeting the Hygro-P2A-mCherry polycistronic construct. Editing efficiency was measured as the loss of mCherry fluorescence per sample, compared to non-transfected control. Error bars indicate the standard deviation of triplicates. The cr-Hygro-3 (AsCas12a) and sg-Hygro-1 (Cas9, dual-guide system) were chosen for further CRISPR-Cas and Acr assays.

Figure 6—figure supplement 5. Phylogenetic reconstruction for Cas12a orthologs.

(A) Phylogenetic reconstruction of Cas12a orthologs relative to TnpB using the full-length Cas12a sequence or (B) the Cas12a RuvC alone. LbCas12a (teal) and AsCas12a (purple) are indicated along with other representative orthologs (bold). The presence of the additional sequence is denoted by a yellow circle to the right of the tree. The size of the Cas12a protein is shown as a gray bar adjacent to the relevant Cas12a ortholog.