Clustered regularly interspaced short palindromic repeats (CRISPR) are derived from a bacterial and archaeal adaptive immune system. The core enzymes of CRISPR are RNA-guided endonucleases that sequence-specifically cleave foreign double-stranded DNA. Improving and controling the properties of the CRISPR system is a crucial step in advancing the therapeutic potential of CRISPR technology. Several classes of these enzymes exist and are being adapted for biotechnology, such as genome engineering. Cas12a (Cpf1) is a Type V CRISPR-associated (Cas) enzyme that naturally uses only one guide RNA, in contrast to Type II CRISPR-Cas9 enzymes. Thus, Cpf1 may represent a simpler, more practical tool for applications such as gene editing and therapeutics. This dissertation comprises four related studies in this area. To better understand the functional requirements for Cpf1-crRNA interaction and develop modified crRNAs suitable for synthetic biology and therapeutic applications, the first study performed nucleotide substitutions in the crRNA. It focused on the protein-interaction motif of the crRNA by incorporating base changes at the 2ʹ position that alter hydrogen-bonding capacity, sugar pucker, and flexibility. DNA substitutions in RNA can probe the importance of A-form structure, 2ʹ-hydroxyl contacts, and conformational constraints within RNA-guided enzymes. In addition, Chemical modifications include 2'-deoxy, 2'-fluoro, 2'- deoxy-arabinonucleic acid, and oxepane. Our study discovered that 2'-fluoro maintains the A-form structure and is compatible with AsCpf1 activity. Biochemical endonuclease activity, gene editing efficiency, Cpf1 binding affinity, and ribonucleoprotein stability were used to assess the tolerance and effects of modification. Characterizing structure-function requirements for Cpf1-crRNA interaction will facilitate better design and tuning of Cpf1 enzymes. The second study established a FRET-based assay in collaboration with a computational collaborator to identify small molecule inhibitors predicted by virtual docking and simulations. This study aims to lay the foundation for efficient, safe implementation of CRISPR-Cpf1. The third study used chemically modified Cas9-guide RNAs to offset known weaknesses of CRISPRi. It takes advantage of the high binding affinity and nuclease resistance of modified guides to potentially reduce the required components for CRISPRi.
| Identifer | oai:union.ndltd.org:siu.edu/oai:opensiuc.lib.siu.edu:dissertations-2835 |
| Date | 01 September 2020 |
| Creators | ageely, Eman |
| Publisher | OpenSIUC |
| Source Sets | Southern Illinois University Carbondale |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations |
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