Structural insight into multistage inhibition of CRISPR-Cas12a by AcrVA4

Abstract

Prokaryotes possess CRISPR-Cas systems to exclude parasitic predators, such as phages and mobile genetic elements (MGEs). These predators, in turn, encode anti-CRISPR (Acr) proteins to evade the CRISPR-Cas immunity. Recently, AcrVA4, an Acr protein inhibiting the CRISPR-Cas12a system, was shown to diminish Lachnospiraceae bacterium Cas12a (LbCas12a)-mediated genome editing in human cells, but the underlying mechanisms remain elusive. Here we report the cryo-EM structures of AcrVA4 bound to CRISPR RNA (crRNA)-loaded LbCas12a and found AcrVA4 could inhibit LbCas12a at several stages of the CRISPR-Cas working pathway, different from other characterized type I/II Acr inhibitors which target only 1 stage. First, it locks the conformation of the LbCas12a-crRNA complex to prevent target DNA-crRNA hybridization. Second, it interacts with the LbCas12a-crRNA-dsDNA complex to release the bound DNA before cleavage. Third, AcrVA4 binds the postcleavage LbCas12a complex to possibly block enzyme recycling. These findings highlight the multifunctionality of AcrVA4 and provide clues for developing regulatory genome-editing tools.

Diversity of molecular mechanisms used by anti-CRISPR proteins: the tip of an iceberg?

“…To date, inhibition of target binding is the prevalent strategy. Thirteen anti-CRISPR proteins interfere with target recognition and binding (type I-F AcrIF1, AcrIF2 and AcrIF10 [34][35][36][37]; type II-A AcrIIA2, AcrIIA4, AcrIIA5 and AcrIIA6 [38][39][40][41][42][43][44][45][46][47]; type II-C AcrIIC3, AcrIIC4 and AcrIIC5 [48][49][50][51]; type V-A AcrVA1, AcrV4A and AcrVA5 [52][53][54][55][56][57][58]), while only five-block target cleavage (type I-E AcrIE1 [29,59]; type III-B AcrIIIB1 [12]; type I-F AcrIF3 [26,27]; type II-C AcrIIC1 and AcrIIC3 [48][49][50]) ( Figure 1). Given that DNA binding is the ratelimiting step of Cascade and Cas9-mediated interference activities [60,61], altering this step is, therefore, an efficient way to inactivate CRISPR-Cas interference.…”

“…AcrIIA6 and AcrVA4 both function as dimers that tightly associate with a mixed protein-RNA region distinct from the DNA-binding crevasse and catalytic domains [47,54,57,58]. However, regions of both subunits that compose the AcrIIA6 dimer form each binding interface, while every subunit of the AcrV4A dimer contains one binding interface ( Figure 2B).…”

“…Additionally, AcrVA4 can also associate with DNA-bound effector complexes. When it binds to the LbCas12a-crRNA-dsDNA complex with a complete R-loop, it induces the release of the bound DNA before cleavage [54,58]. When it binds to the post-cleavage Cas12a-crRNA-dsDNA complex, it likely blocks the recycling of the enzyme [58].…”

Types I and V Anti-CRISPR Proteins: From Phage Defense to Eukaryotic Synthetic Gene Circuits

“…In the Cas12a-crRNA-AcrVA4 complex, the β4-β5 loop forms a tight salt bridge with BH that locks the BH itself into the AcrVA4 structure. Hence, Arg887 rotation (and the corresponding conformational change in the crRNA-DNA heteroduplex formation) can no longer take place (Knott et al, 2019a;Peng et al, 2019;Zhang H. et al, 2019). Furthermore, the interaction between AcrVA4 β4-β5 loop and Cas12a REC lobe obstructs the REC2 movement necessary for crRNA-DNA heteroduplex propagation (Peng et al, 2019).…”

“…AcrVA4 can also dislodge Cas12a-crRNA from the substrate DNA and, therefore, prevent target DNA cleavage by the CRISPR-Cas12a system. Only high concentrations of AcrVA4 make possible the release of the target DNA from the complex with Cas12a-crRNA (Knott et al, 2019b;Peng et al, 2019). Besides, AcrVA4 takes part in post-cleavage inhibition.…”