Unveiling the structural determinants for enhanced specificity in CRISPR/Cas9 for genome editing

Abstract

CRISPR/Cas, a recently discovered genome-editing method depends on a single protein (Cas9) and non-coding RNA for gene-editing, which makes it simple, more rapid, versatile, efficient and manipulatable. Despite the high-efficiency and user-friendliness of CRISPR/Cas9, its applications are limited by various factors which includes large size of Cas9 (difficult in delivery), recognition to a specific PAM (limiting its effectiveness), differential specificity and sensitivity of Cas variants and introduction of random off target mutations at sequences similar to those of target genes, major concern. An ortholog of Cas9 from Francisella novicida (FnCas9), was shown to have very low non-specific editing compared to SpCas9. The cleavage and recognition mechanism of FnCas9 as well as the reasons for its increased specificity have not yet been fully investigated, whereas SpCas9 (derived from Streptococcus pyogenes bacteria) has been the subject of much research. Questions pertaining to the molecular interplay for the interactions and a comparison between the two orthologs can provide us insights into the elusive future of designing customized Cas9 molecules. This study provides a comprehensive examination of the structural and dynamic characteristics of SpCas9 and FnCas9, two prominent CRISPR/Cas9 orthologs, to elucidate the molecular mechanisms that govern their specificity, efficiency, and substrate versatility in genome editing. Utilizing molecular dynamics (MD) simulations, we compared the apo and gRNA-bound states of these proteins, revealing distinct conformational shifts and interactions that significantly influence gRNA binding and cleavage activity. FnCas9 demonstrated superior stability in gRNA binding attributed to dynamic domain rearrangements and specific residue interactions, particularly in the bridge-helix and REC3 domain. To further investigate specificity, accelerated MD simulations combined with machine learning techniques were employed to analyze RNA:DNA hybrid mismatches, revealing that FnCas9 maintains structural integrity in the presence of PAM-distal mismatches, unlike SpCas9. Enhanced interactions within the REC3 domain and allosteric communication pathways bypassing the REC2 domain were identified as critical factors in base-pair mismatch recognition and efficient gene-editing. Additionally, structural analyses of engineered FnCas9 variants (en1, en15, en31) highlighted how specific mutations and domain modifications impact cleavage efficiency and specificity. The en31 variant exhibited distinct domain dynamics supporting better base-pair mismatch discrimination and adaptability for broader genome-editing targets due to its. Finally, the study compared FnCas9’s interaction with RNA (tRNA) and DNA (tDNA) substrates, revealing unique interaction networks that suggest differing binding dynamics. While tDNA (target DNA) showed stronger binding affinity overall, tRNA (target RNA) binding induced greater conformational flexibility in key domains. These findings advance our understanding of the structural determinants of Cas9 function and provide actionable insights for engineering high-fidelity Cas9 variants with enhanced precision and substrate compatibility. Future directions include mechanistic studies on DNA-bound ternary complexes and the development of optimized Cas9 systems for RNA editing applications, contributing to the ongoing evolution of genome editing technologies.