RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2

Jack P. K. Bravo, Thomson Hallmark, Bronson Naegle, Chase L. Beisel, Ryan N. Jackson () and David W. Taylor ()
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Jack P. K. Bravo: University of Texas at Austin
Thomson Hallmark: Utah State University
Bronson Naegle: Utah State University
Chase L. Beisel: Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Centre for Infection Research (HZI)
Ryan N. Jackson: Utah State University
David W. Taylor: University of Texas at Austin

Nature, 2023, vol. 613, issue 7944, 582-587

Abstract: Abstract Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA following recognition of a complementary RNA target, culminating in abortive infection1. 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 double-stranded DNA degradation and in vivo immune system function. Our work provides a structural basis for this mechanism of abortive infection to achieve population-level immunity, which can be leveraged to create rational mutants that degrade a spectrum of collateral substrates.

Date: 2023
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DOI: 10.1038/s41586-022-05560-w

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