Bio-orthogonal chemistry-based strategy to Turn-OFF CRISPR-Cas9 activity in solution and live cells

Abstract The CRISPR–Cas9 system has become a widely used gene-editing tool. Here, we present a new method for small-molecule control of CRISPR-Cas9 using bio-orthogonal chemistry between tetrazine (Tz) and trans-cyclooctene (TCO). We carried out molecular modeling studies and identified a unique position on single guide RNA (sgRNA) that can be site-specifically tagged with Tz without disrupting its activity. We also synthesized a series of TCO-modified CRISPR suppressors. When exogenously added, they bind to the Tz-tagged sgRNA, perturb the system, and drastically reduce the nuclease activity. The most successful suppressor is a TCO-modified six-amino acid-long cell-penetrating peptide, which shows excellent cell permeability. We showed that our method to control CRISPR-Cas9 nuclease activity is general by applying it to three different sgRNAs. We also showed that our method works in solution, as well as live HEK293 cells. We utilized flow cytometry to demonstrate the inactivation of the CRISPR-Cas9 system targeting GFP. Lastly, we showed the therapeutic potential of our method by targeting vascular endothelial growth factor A (VEGFA). Overall, the described method enables robust and minimally perturbing control of CRISPR–Cas9 nuclease activity using TCO suppressors to regulate Tz-modified sgRNA, targeting various genes in vitro and in live cells, offering a broadly applicable platform for controlled gene editing.