Knight, A. L. | bioRxiv Science

bioRxiv2026

Orthosteric and allosteric effects of anti-CRISPR II-C1 inhibition on GeoCas9 from integrated structural biophysics

Anti-CRISPRs (Acrs) are small protein inhibitors of CRISPR-Cas effectors that originate from the translated genetic material of bacteriophage. Harnessing the natural ability of Acrs to bind and disrupt CRISPR-Cas editing can provide enhanced spatiotemporal control of gene editing. Recent studies have revealed diverse structures and functions of Acrs, however, atomistic studies of the specific molecular mechanisms behind Acr inhibition are lacking. Here, we reveal how structure, function, and dynamics govern AcrIIC1 inhibition of Cas9 from G. stearothermophilus (GeoCas9) via its HNH nuclease domain. An X-ray crystal structure of the GeoHNH-AcrIIC1complex reveals a conserved binding interface at the catalytic site and disruption of crucial electrostatic contacts known to modulate the thermostability of GeoCas9. AcrIIC1 binding also rewires the intrinsic dynamics of the GeoHNH domain, stimulates millisecond motions that are absent from the unliganded nuclease, and attenuates the guide RNA affinity of GeoCas9. Subsequent AcrIIC1 mutations in residues at its crystallographic binding interface uncouple Acr binding from inhibition, providing new insight into mechanism by which AcrIIC1 acts on GeoCas9.

biophysics↗

bioRxiv2024

Atomistic Tuning of the GeoCas9 Recognition Lobe Modulates Allosteric Motions and Guide RNA Interactions

The intuitive manipulation of specific amino acids to alter the activity or specificity of CRISPR-Cas9 has been a topic of great interest. As a large multi-domain RNA-guided endonuclease, the intricate molecular crosstalk within the Cas9 protein hinges on its conformational dynamics, but a comprehensive understanding of the extent and timescale of the motions that drive its allosteric function and association with nucleic acids remains elusive. Here, we investigated the structure and multi-timescale molecular motions of the recognition (Rec) lobe of GeoCas9, a thermophilic Cas9 from Geobacillus stearothermophilus. Our results provide new atomic details about the GeoRec subdomains (GeoRec1, GeoRec2) and the full-length domain in solution. Two rationally designed mutants, K267E and R332A, enhanced and redistributed micro-millisecond flexibility throughout GeoRec, and NMR studies of the interaction between GeoRec and its guide RNA showed that mutations reduced this affinity and the stability of the ribonucleoprotein complex. Despite measured biophysical differences due to the mutations, DNA cleavage assays reveal no functional differences in on-target activity, and similar specificity. These data suggest that guide RNA interactions can be tuned at the biophysical level in the absence of major functional losses but also raise questions about the underlying mechanism of GeoCas9, since analogous single-point mutations have significantly impacted on- and off-target DNA editing in mesophilic S. pyogenes Cas9. A K267E/R332A double mutant did also did not enhance GeoCas9 specificity, highlighting the robust tolerance of mutations to the Rec lobe of GeoCas9 and species-dependent complexity of Rec across Cas9 paralogs. Ultimately, this work provides an avenue by which to modulate the structure, motion, and guide RNA interactions at the level of the Rec lobe of GeoCas9, setting the stage for future studies of GeoCas9 variants and their effect on its allosteric mechanism.

biophysics↗