Tailoring CRISPR/Cas specificity using chemically modified guide RNAs

• Author / Creator

  Cromwell, Christopher Robert

• The ability to reliably target specific sequences of interest is a critical component of any gene editing technology. While the discovery of CRISPR-Cas systems have fundamentally simplified the way in which genomic material may be manipulated, the presence of off-target activity at undesired loci still remains a concern, especially with regards to human clinical applications. Conversely, the high levels of polymorphic sequences present within the natural population also present a unique challenge to the broader application of CRISPR-based therapeutics, as high levels of specificity may predispose some individuals to therapy failure.

  Here, I describe how a variety of chemically modified nucleotides may be incorporated into the sequence-specific spacer region of the crRNA of either Cas9 or Cas12a, in order to modulate specificity. Initially, I demonstrated that chemically modified nucleotides (such as bridged nucleic acids and locked nucleic acids) are an effective strategy for broadly enhancing the specificity of Cas9 cleavage both in vitro, and in cells. In addition to providing design considerations for the effective use of BNA/LNA-modified crRNAs, I also identified a potential biochemical mechanism for the observed specificity improvements through smFRET experiments with collaborators. Building upon these discoveries, I expanded the scope of my work to include several additional chemically modified nucleic acids (or xeno nucleic acids; XNAs). Our lab hypothesized that while BNA/LNA incorporation lead to higher specificities, we may also be able to identify novel modifications with even greater fidelities. Among the XNA modified crRNAs (crXNAs) tested, I describe several crXNAs capable of imparting high levels of cleavage specificity to the Cas9 system. I also show that while different crXNAs may display similar levels of off-target discrimination overall, each crXNA has a unique specificity profile regarding the off-target sequences they are able to effect.

  Given our success with crXNAs, we also investigated the ability of chemically modified nucleotides to impart properties to CRISPR-based systems independent of enhancing specificity. With collaborators, I demonstrated that through the incorporation of universal bases into the spacer sequences of either Cas9 or Cas12a crRNAs, we are able to target multiple polymorphic sequences without loss of overall specificity. Alongside these collaborators, I helped to demonstrate the applicability of this technology to the targeting of genes with high levels of polymorphisms, as well as to the in vitro diagnostic platform known as DETECTR, for the detection of multiple HIV- 1 variant sequences.

  In summary, the work presented herein displays the versatility of chemically modified crRNAs for augmenting the capability of CRISPR-based technologies with novel properties. These findings outline a framework for the identification and characterization of chemically modified guide RNAs, introducing a versatile tool for future CRISPR-based research.

• Subjects / Keywords

  • CRISPR
  • Gene editing
  • Cas9
  • Chemically modified nucleotide
  • CRISPR specificity

• Graduation date

  Fall 2022

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-6kv4-qp74

• License

  This thesis is made available by the University of Alberta Library 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.