Large-scale structural rearrangements unleash indiscriminate nuclease activity of CRISPR-Cas12a2

Abstract

Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided degradation of non-specific single-stranded (ss)RNA, ssDNA and double-stranded (ds)DNA upon recognition of a complementary RNA target, culminating in abortive infection (Dmytrenko 2022). Here, we report structures of Cas12a2 in binary, ternary, and quaternary complexes to reveal a complete activation pathway. Our structures reveal that Cas12a2 is autoinhibited until binding a cognate RNA target, which exposes the RuvC active site within a large, positively charged cleft. Double-stranded DNA substrates are captured through duplex distortion and local melting, stabilized by pairs of "aromatic clamp" residues that are crucial for dsDNA degradation and in vivo immune system function. Our work provides a structural basis for this unprecedented mechanism of abortive infection to achieve population-level immunity, which can be leveraged to create rational mutants that degrade a spectrum of collateral substrates.

Cas12a2 elicits abortive infection via RNA-triggered destruction of double-stranded DNA

“…These results demonstrate that RNA targeting by SuCas12a2 causes dsDNA damage of the bacterial chromosome that in turn induces the SOS response and Abi in bacteria, reflecting a distinct mechanism of immunity that relies on indiscriminate dsDNase activity. Consistent with this observation, recent cryo-EM structures reveal Cas12a2 binds and cuts dsDNA with a mechanism completely distinct from all other CRISPR associated nucleases, while structure-guided mutants with impaired in vitro collateral dsDNase but not ssRNase and ssDNase activities abolished in vivo defense activity 20 .…”

“…Investigating the underlying molecular basis of target recognition and activation of collateral cleavage by Cas12a2 could reveal new mechanisms employed by CRISPR nucleases for discriminating self from non-self targets. Recent cryo-EM structures of Cas12a2 at stages of RNA-targeting and collateral dsDNA capture are already fulfilling this need 20 .…”

“…2). Considering their original classification as well as our phylogenetic data and structural results 20 , we named these distinct type V nucleases Cas12a2.…”

An Alpha-helical Lid Guides the Target DNA toward Catalysis in CRISPR-Cas12a

“…Structural studies showed that this ⍺-helical lid occludes the RuvC catalytic core in the binary RNA-bound Cas12a complex and opens for the accommodation of the NTS within the catalytic site. 12,30 Multi-μs long MD simulations of Cas12a after NTS cleavage show that the ⍺-helical lid establishes extensive interactions with the crRNA:TS hybrid in its open conformation (Fig. 2a-b).…”