Bio-Orthogonal Chemistry-Based Strategy to Turn-On and Turn-Off CRISPR-Cas9 Gene Editing in Solution and in Live Cells

The CRISPR–Cas9 system is a widely popular tool for genome engineering. There is a strong interest in developing tools for temporal control of CRISPR-Cas9 activity to address some of the challenges and to broaden the scope of potential applications. In this thesis I describe two biorthogonal based approaches to either Turn-ON and Turn-OFF CRISPR-Cas9 gene editing. In first project work I describe a bio-orthogonal chemistry-based approach to Turn-ON Cas9 nuclease activity with temporal precision. We report a TCO-acylimidazole reagent that acylates 2′-OH groups of RNA. Poly-acylation (“cloaking”) of RNA was optimized in vitro using a model 18-nt oligonucleotide, as well as CRISPR single guide RNA (sgRNA). Two hours of treatment completely inactivated sgRNA for Cas9-assisted DNA cleavage. Nuclease activity was restored upon addition of tetrazine that removes the TCO moieties via a two-step process (“uncloaking”). The approach was applied to target the GFP gene in live HEK293 cells. GFP expression was analyzed by flow cytometry. In the future, we anticipate that our approach will be useful in the field of developmental biology, by enabling investigation of genes of interest at different stages of organism’s development. In second part of thesis, I present a new method for small-molecule control to Turn-OFF CRISPR-Cas9 using bio-orthogonal chemistry between tetrazine (Tz) and trans-cyclooctene (TCO). I 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. I also synthesized a series of TCO-modified CRISPR suppressors. When exogenously added, they click 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. I 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 temporal control of CRISPR-Cas9 targeting GFP. Lastly, we showed the therapeutic potential of our method by targeting vascular endothelial growth factor A (VEGFA).