Innovative Chemical Strategies for Advanced CRISPR Modulation.

ConspectusOver the past decade, RNA-guided gene editing technologies, particularly those derived from CRISPR systems, have revolutionized life sciences and opened unprecedented opportunities for therapeutic innovation. Despite their transformative potential, achieving precise control over the activity and specificity of these molecular tools remains a formidable challenge, requiring advanced and innovative regulatory strategies. We and others have developed new approaches that integrate chemical ingenuity with bioorthogonal techniques to achieve remarkable precision in CRISPR regulation. One key innovation lies in the chemical modulation of guide RNA (gRNA), significantly expanding the CRISPR toolkit. Strategies such as CRISPR-ON and CRISPR-OFF switches rely on selective chemical masking and demasking of gRNA. These approaches use either bulky chemical groups to preemptively mask RNA or minor, less obstructive groups to fine-tune its function, followed by bioorthogonal reactions to restore or suppress activity. These methodologies have proven to be pivotal for controlled gene editing and expression, addressing the challenges of precision, reversibility, and dynamic regulation.Parallel to these advances, the development of mesoporous metal-organic frameworks (MOFs) has emerged as a promising solution for RNA deprotection and activation. By serving as catalytic tools, MOFs enhance the versatility and efficiency of CRISPR systems, pushing their applications beyond the conventional boundaries. In addition, the synthesis of novel small molecules for regulating CRISPR-Cas9 activity marks a critical milestone in the evolution of gene therapy protocols. Innovative RNA structural control strategies have also emerged, particularly through the engineering of G-quadruplex (G4) motifs and G-G mismatches. These methods exploit the structural propensities of engineered gRNAs, employing small-molecule ligands to induce specific conformational changes that modulate the CRISPR activity. Whether stabilizing G4 formation or promoting G-G mismatches, these strategies demonstrate the precision and sophistication required for the molecular-level control of gene editing.Further enhancing these innovations, techniques like host-guest chemistry and conditional diacylation cross-linking have been developed to directly alter gRNA structure and function. These approaches provide nuanced, reversible, and safe control over CRISPR systems, advancing both the precision and reliability of gene editing technologies. In conclusion, this body of work highlights the convergence of chemistry, materials science, and molecular biology to create integrative solutions for gene editing. By combination of bioorthogonal chemistry, RNA engineering, and advanced materials, these advancements offer unprecedented accuracy and control for both fundamental research and therapeutic applications. These innovations not only advance genetic research but also contribute to developing safer and more effective gene editing strategies, moving us closer to realizing the full potential of these technologies.