Enhancing CRISPR-Cas9 Precision with Small Molecule Modulators: Mechanistic Insights and Novel Discovery

• Applicant: Dr. Xinlai Cheng
• Subject Area: Pharmacy ; Biochemistry
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 491581972

CRISPR-Cas9 genome editing technology has profoundly transformed biology and medicine, offering unprecedented potential for precise genetic manipulation. Yet its clinical application remains limited by unintended off-target effects, immunogenic risks, and high costs, highlighting the urgent need for strategies that enhance specificity, safety, and affordability. During the current funding period, we established robust biochemical, biophysical, and cellular assays to identify and characterize small-molecule modulators of Cas9. Using our optimized thermal shift screening platform, we identified several potent Cas9 modulators. In combination of AI-driven modeling and experimental validation, including a suite of biochemical assays, structural biology, and cellular reporter systems we characterized that multiple plant-derived compounds bind specifically to the Cas9 bridge-helix/REC1 interface, effectively blocking off-target DNA binding while preserving on-target editing. We also identified that certain FDA-approved drugs stabilized Cas9 by binding the REC3 domain, significantly increasing on-target specificity. The results were disseminated through several presentations at renowned scientific conferences, with manuscripts and patent applications currently under preparation. In the proposed renewal, we aim to deepen our mechanistic understanding of these modulators and discover additional novel small molecules that further improve Cas9 precision and efficiency. To achieve this, we will pursue two major work packages: (1) detailed structural and functional analyses of leader compounds, using high-resolution crystallography, cryo-electron microscopy, and comprehensive biochemical assays; and (2) expanded high-content screening of enriched natural product and macrocyclic libraries, followed by rigorous multi-step validation. Top candidates from these screenings will undergo systematic characterization, including surface plasmon resonance, electrophoretic mobility shift assays, fluorescence polarization assays, cellular genome-editing assays, and next-generation sequencing-based specificity profiling (Amplicon-Seq, GUIDE-Seq, Circle-Seq). Through these integrated approaches, we expect to generate definitive insights into small molecule-Cas9 interactions, establish clear structure-activity relationships, and identify lead compounds ready for preclinical validation. Ultimately, this renewal project will significantly advance chemical strategies for precision genome editing, paving the way toward safer, more effective, and more accessible CRISPR-based therapies.

• DFG Programme: Research Grants