Applications and Improvements of CRISPR/Cas9 and Cas12a Gene Editing Technologies

• Author / Creator

  Krysler, Amanda

• Background: The CRISPR/Cas9 system has emerged as a revolutionary genetic engineering technology capable of editing various cell types, creating disease models, and more recently, changing human DNA1 . It consists of three pieces: the Cas9 endonuclease, the gRNA, and the DNA target. In short, the Cas9 protein is ‘guided’ by the gRNA to cut a specified DNA target. This system has risen above other genetic engineering tools due to the feasibility of programming the 20-nt sequence of the gRNA to target a complementary DNA sequence. This programmability provides researchers with an easy and efficient way to knockout and study proteins of interest, as demonstrated in Chapter 3 of this thesis. Despite the promise of this new system, there are some major roadblocks preventing easy adaptation to the clinical setting. One of these challenges is the natural variability of our genome, as it limits the researcher's ability to target all versions of a highly polymorphic DNA sequence2 . In Chapter 4, we propose the use of universal bases, ie: bases which can pair with any of the naturally found DNA bases, to target SNPs3 . This would allow for one gRNA to target all four versions of a DNA sequence instead of having to design and deliver four separate gRNAs. This would further expand applications of the CRISPR system, as outlined in Chapter 5. Methods: In Chapter 3, gRNAs were designed to target the early exons of the gene of interest, for each collaborating lab. After the transfection of the desired cell line, the knockout cell populations were screened and sorted with FACs. Each single clone population was then tested for efficient disruption of the genomic DNA and subsequent iii lack of the desired protein. In Chapter 4, gRNAs with universal and degenerate bases were designed to target highly polymorphic genes: HLA and ABO. DNA sequences containing naturally occurring SNPs were synthesized for each gene. In vitro cleavage assays were performed to determine the activity of Cas9 on each DNA sequence using different modified gRNAs. A specificity profile for all the modified gRNAs was then created using an in vitro high through-put assay. The best gRNA from these two assays was further validated by testing in cells. Finally, the best combination and type of modification was applied to an alternative CRISPR system, Cas12a, and used in a detection assay targeting a polymorphic section of the HIV-1 genome. Results: In Chapter 3, 3 different protein knockout cell lines (CRMP2A, FAM120B, and B4GALNT1) were created and fully validated. One final cell line has a predicted 50% knockdown of SF3B4 protein based on the genomic DNA results, however, further protein validation is yet to be performed. In Chapter 4, we show that the addition of universal bases resulted in increased activity at SNP targets which would not have been cut by the current wildtype gRNA. The addition of these bases resulted in selective degeneracy at the DNA positions complementary to the positions of the incorporated modifications in the gRNA. The best gRNA from these experiments contained 3 ribose inosines. Application of these ribose inosines to the Cas12a gRNAs also resulted in successful cleavage of only the relevant SNP targets. This was reflected in the DETECTR4 assay where a gRNA targeting a polymorphic region of the HIV-1 genome was used. Conclusions: As shown in Chapter 3, CRISPR/Cas9 knockout cell lines were successfully created for each desired protein and can now be used in functional studies. These will be used to investigate disease pathology, drug pathways, mitochondrial gene interactions, and lung cancer characteristics. In Chapter 4, we demonstrate that the incorporation of universal bases results in increased cleavage activity of their respective SNP target(s) without losing overall targeting specificity. This was validated using both in vitro and cellular assays and also applied to a detection assay developed for clinical use. Overall, the addition of universal and degenerate bases addresses the problem of targeting the innate diversity of the human and viral genome and introduces additional applications of the CRISPR/Cas9 and Cas12a system for both bench and clinical research. These future applications are described in Chapter 5.

• Subjects / Keywords

  • Gene editing
  • Universal Bases
  • Cas12a
  • CRISPR/Cas9

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-vkv3-e516

• License

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