
GeoCas9の認識ローブ(Rec)の構造、動態、およびガイドRNA結合を原子レベルで調整することで、GeoCas9の機能と特異性を微調整できる。


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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 Geo Cas9, a thermophilic Cas9 from Geobacillus stearothermophilus. Our results provide new atomic details about the Geo Rec subdomains ( Geo Rec1, Geo Rec2) and the full-length domain in solution. Two single-point mutants, K267E and R332A, enhanced and redistributed micro-millisecond flexibility throughout Geo Rec, and NMR studies of the interaction between Geo Rec 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 only modest functional differences in on-target activity, and similar specificity. These data highlight how guide RNA interactions can be tuned in the absence of major functional losses, but also raise questions about the underlying mechanism of Geo Cas9, 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 modestly enhance Geo Cas9 specificity, highlighting the robust evolutionary tolerance of Cas9 and species-dependent complexity. Ultimately, this work provides an avenue by which to modulate the structure, motion, and nucleic acid interactions at the level of the Rec lobe of Geo Cas9, setting the stage for future studies of Geo Cas9 variants and their effect on its allosteric mechanism.

### Competing Interest Statement

The authors have declared no competing interest.

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

熱安定性の高いgeocas9の認識ローブの原子レベルでのチューニングが-アロステリック運動とガイドrna相互作用を変調させる


熱安定性の高いGeoCas9の認識ローブの原子レベルでのチューニングが、アロステリック運動とガイドRNA相互作用を変調させる
