Cas12a (Cpf1) is a representative type V-A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) effector RNA-guided DNA endonuclease used widely for genome editing. Identification of Cas12a inhibitors is important for regulating gene editing, enhancing genome editing specificity, and safety for human therapeutics. This study used a fluorescence-based assay to screen diverse drug libraries at a core facility for potential small molecule candidates that can inhibit AsCas12a endonuclease activities. Further validation of the major hit compounds revealed that these small molecules inhibit Cas12a in vitro DNA cis and trans cleavage activities as well as gene editing in cells. IC50 values obtained from gene editing inhibition were even lower than primary screening reported IC50. We determined the impact of the small molecules on the thermal stability of Cas12a, possible binding sites, and binding affinity (Kd) using thermal denaturation experiments. Enzyme kinetics studies were used to investigate the effect of the inhibitors on ribonucleoprotein complex formation. The discovered molecules create a tool for achieving safer applications of CRISPR-Cas12a in biotechnology and therapeutics.
| Identifer | oai:union.ndltd.org:siu.edu/oai:opensiuc.lib.siu.edu:theses-4064 |
| Date | 01 December 2022 |
| Creators | Yinusa, Abadat |
| Publisher | OpenSIUC |
| Source Sets | Southern Illinois University Carbondale |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses |
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