Discovery and Characterization of Small-Molecule Inhibitors of CRISPR/Cas13a

• Author / Creator

  Reformat, Filip M

• Background: Clustered Regularly Interspaced Palindromic Repeats, or CRISPR, is an array of DNA sequences found in bacteria that confer immunity against phage. Sequences within the CRISPR array are transcribed into CRISPR RNA (crRNA), which hybridize with CRISPR associated proteins (Cas) to search for and cleave nucleic acids of the invading phage. A variety of CRISPR systems have been discovered and characterized, one member of this family, the DNA targeting enzyme Cas9, has had a significant impact in the field of biology and medicine after being adapted into a robust and intuitive gene-editing tool. Another recently discovered member of the CRISPR family called Cas13 is unique in its ability to target and cleave RNA rather than DNA. CRISPR/Cas13 has been used to knock down RNA transcripts in a variety of organisms and has been adapted into a diverse range of tools. Despite its wide range of applications, Cas13 has its limitations: toxicity due to prolonged RNA knockdown, and potential for off-target effects. Inhibitory small-molecule compounds could address these issues by imparting temporal control over Cas13. This dissertation presents my main research project: discovery and characterization of small-molecule inhibitors of Cas13a.

  Methods: We adapted and employed a FAM (6-Carboxyfluorescein) cleavage assay, which utilizes a fluorophore/quencher reporter RNA molecule, to screen for Cas13a inhibitors. In this assay, cleavage of the reporter RNA removes the quencher leading to a fluorescent signal. If a compound inhibits Cas13a then there will be no detectable fluorescent signal. Over 13000 compounds were screened using this assay. The most effective inhibitors were identified and validated with the FAM assay. Furthermore, they were subjected to a quenching assay, a redox cycling test via the Amplex® Red assay, and a non-fluorophore based gel cleavage assay to confirm genuine inhibition. The compounds that passed these tests had their IC50s and mechanism of inhibition determined.

  Results: Using the aforementioned approach, we found nine compounds that were able to inhibit Cas13a. The IC50 of these compounds ranged from 1 µM to 3 µM. Most of the compounds were either non-competitive or competitive inhibitors, and initial cell data showed that most compounds were non-toxic.

  Conclusion: We have demonstrated the ability to use a fluorescent assay in a high-throughput approach to find small molecule inhibitors of Cas13a. A total of nine chemical compounds have been validated and characterized. Additional experiments need to be performed to examine the compounds’ effect on RNA binding and their ability to inhibit Cas13a in cells. If successful, these compounds could prove to be useful in laboratory work and clinical trials to limit the duration of Cas13a activity.

• Subjects / Keywords

  • CRISPR/Cas13
  • CRISPR
  • Cas13
  • Cas13 Inhibitors
  • Drug Discovery

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-asn3-t576

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.