Development and Implementation of a Novel CRISPR/Cas9 System to Investigate Translational Repression Mechanisms in Plasmodium yoelii.
Open Access
- Author:
- Walker, Michael
- Graduate Program:
- Molecular, Cellular and Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 23, 2021
- Committee Members:
- Scott E. Lindner, Dissertation Advisor/Co-Advisor
Scott E. Lindner, Committee Chair/Co-Chair
Melissa Rolls, Committee Member
Manuel Llinas, Committee Member
Howard M Salis, Outside Member
Maciej F Boni, Committee Member
Melissa Rolls, Program Head/Chair - Keywords:
- Malaria
CRISPR
Plasmodium
yoelii
TRIBE
Translational Repression
RNA
DNA
RGR - Abstract:
- Malaria remains a major global health issue, affecting millions and killing hundreds of thousands of people annually. Efforts to break the transmission cycle of the causative agent, the Plasmodium parasite, and to cure those that are afflicted, rely upon functional characterization of genes essential to the parasite’s growth and development. These studies are often based upon manipulations of the parasite genome to disrupt or modify a gene-of-interest to understand its importance and function. However, these approaches can be limited by the availability of sufficient selectable markers, by the time required to generate transgenic parasites, and by exogenous sequences left behind. To address these limitations, I have developed CRISPR-RGR, a Streptococcus pyogenes (Sp)Cas9-based gene editing system for Plasmodium that utilizes a Ribozyme-Guide-Ribozyme (RGR) single-guide RNA (sgRNA) expression strategy with RNA polymerase II promoters. Using rodent-infectious Plasmodium yoelii, I have demonstrated that both gene disruptions and coding sequence insertions are efficiently generated, the number of sgRNA targets can affect the outcome of genome repair, and that CRISPR-RGR can be used for CRISPR interference (CRISPRi). I then used this system to interrogate translational repression mechanisms in salivary gland sporozoites, the life stage of the parasite that is transmitted from mosquito to mammal during a biting event. By genetically altering a gene encoding the transcript pyuis4, I demonstrated that its translational repression is controlled by something within its ORF. But, this regulatory mechanism does not appear to be dependent upon the two putative Puf-binding elements present in the pyuis4 ORF. Lastly, I show that PyPuf2, an RNA-binding protein known to play a role in translational repression, associates with distinct transcripts in salivary gland sporozoites. However, based on my observations with pyuis4, this association may not be dependent on the putative Puf-binding elements. Combined, this dissertation discusses the development of a robust and effective tool that aided in the investigation of processes essential to the infectivity of the Plasmodium parasite.