Structural and Dynamic Impacts of Single-atom Disruptions to Guide RNA Interactions within the Recognition Lobe of Geobacillus stearothermophilus Cas9 DOI Open Access
Helen B. Belato,

Alexa L Knight,

Alexandra M. D’Ordine

и другие.

Опубликована: Март 27, 2025

The intuitive manipulation of specific amino acids to alter the activity or specificity CRISPR-Cas9 has been a topic great interest. As large multi-domain RNA-guided endonuclease, intricate molecular crosstalk within Cas9 protein hinges on its conformational dynamics, but comprehensive understanding extent and timescale motions that drive allosteric function association with nucleic remains elusive. Here, we investigated structure multi-timescale recognition (Rec) lobe Geo Cas9, thermophilic from Geobacillus stearothermophilus. Our results provide new atomic details about Rec subdomains ( Rec1, Rec2) full-length domain in solution. Two rationally designed mutants, K267E R332A, enhanced redistributed micro-millisecond flexibility throughout Rec, NMR studies interaction between guide RNA showed mutations reduced this affinity stability ribonucleoprotein complex.Despite measured biophysical differences due mutations, DNA cleavage assays reveal no functional on-target activity, similar specificity. These data suggest interactions can be tuned at level absence major losses, also raise questions underlying mechanism since analogous single-point have significantly impacted on- off-target editing mesophilic S. pyogenes Cas9. A K267E/R332A double mutant did not enhance specificity, highlighting robust tolerance species-dependent complexity across paralogs. Ultimately, work provides an avenue by which modulate structure, motion, setting stage for future variants their effect mechanism.

Язык: Английский

Structural and Dynamic Impacts of Single-atom Disruptions to Guide RNA Interactions within the Recognition Lobe of Geobacillus stearothermophilus Cas9 DOI Open Access
Helen B. Belato,

Alexa L Knight,

Alexandra M. D’Ordine

и другие.

Опубликована: Март 27, 2025

The intuitive manipulation of specific amino acids to alter the activity or specificity CRISPR-Cas9 has been a topic great interest. As large multi-domain RNA-guided endonuclease, intricate molecular crosstalk within Cas9 protein hinges on its conformational dynamics, but comprehensive understanding extent and timescale motions that drive allosteric function association with nucleic remains elusive. Here, we investigated structure multi-timescale recognition (Rec) lobe Geo Cas9, thermophilic from Geobacillus stearothermophilus. Our results provide new atomic details about Rec subdomains ( Rec1, Rec2) full-length domain in solution. Two rationally designed mutants, K267E R332A, enhanced redistributed micro-millisecond flexibility throughout Rec, NMR studies interaction between guide RNA showed mutations reduced this affinity stability ribonucleoprotein complex.Despite measured biophysical differences due mutations, DNA cleavage assays reveal no functional on-target activity, similar specificity. These data suggest interactions can be tuned at level absence major losses, also raise questions underlying mechanism since analogous single-point have significantly impacted on- off-target editing mesophilic S. pyogenes Cas9. A K267E/R332A double mutant did not enhance specificity, highlighting robust tolerance species-dependent complexity across paralogs. Ultimately, work provides an avenue by which modulate structure, motion, setting stage for future variants their effect mechanism.

Язык: Английский

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