Date of Award

8-2017

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Chemistry and Biochemistry

Thesis Sponsor/Dissertation Chair/Project Chair

John Siekierka

Committee Member

Nina Goodey

Committee Member

Mitchell Sitnick

Abstract

Leishmaniasis is a parasitic Neglected Tropical Disease (NTD) that affects mostly poor countries of the third word due to their limited access to clean water and health care. The disease is transmitted through the bite of a sand fly of the genus Phlebotomus in the Old World, and of the genus Lutzomyia in the New World. Historically, NTDs have not been given adequate attention or research funding and finding curative treatments has been slow. However, the fact that the insect vector and the parasite can live and thrive in any ecological system in the world, and under the current circumstances of the world's worst refugee crisis playing a major role in the geographical distribution of the disease, leishmaniasis has been given more attention. Leishmaniasis is manifested in cutaneous or mucosal forms causing scan-ing, disfiguration, bleeding and breathing difficulties, or in a visceral form that causes liver and spleen malfunction, a compromised immune system and death if not treated. Currently, there is no curative treatment or vaccination for leishmaniasis. The available treatments aim to reduce morbidity from the cutaneous and mucosal leishmaniasis, and mortality from the visceral fonn. Finding new medicines for leishmaniasis requires identifying new drug targets, hence, gene function studies are essential for revealing potential specific drug targets in this parasite.

Although the whole genomes of various leishmania species have been sequenced, very little is known about the gene functions due to the absence of an RNAi pathway in the parasite and the difficulty of achieving homozygous knock out (KO) by the traditional homologous recombination methodology in a diploid organism like leishmania. The new CRISPR/Cas9 technology for targeted gene editing holds great promise for easier study of gene functions in leishmania, hence allowing identification of new drug targets.

Very few studies were done on applying CRISPR/Cas9 in Leishmania. Therefore, we attempted to adapt the CRISPR/Cas9 system to Leishmania donovani for a proof of concept. In this research we describe a new culture medium not typically used in the literature for L.donovani that allows optimal growth of promastigotes in vitro. Also we describe protocols for genomic DNA extraction as well as oligonucleotide annealing and cloning conditions adjusted specifically for the generation of gRNA expression plasmids for leishmania. Furthermore, we established a stable Leishmania cell line expressing Cas9, and set up the basic strategy for applying the CRISPR/Cas9 gene knockout technology in L. donovani. After specifically targeting the ODC (omithine decarboxylase) gene for KO, we found by DNA sequence analysis that no Insertion or Deletion (lnDel) mutations occurred at the targeted site. This finding indicates that although the Cas9 enzyme is likely to scan and cut the doublestranded DNA frequently at the targeted site, the double strand breaks are being repaired accurately each time by an error free repair machinery such as Homologous Directed Repair. Thus, we propose as specific future direction the use of HDR for interruption of the gene open reading frame instead of relying on simple Non-Homologous End Joining for creating random InDels upon repairing the double stranded break by Cas9. Overall, this study paved the way for adapting the CRISPR/Cas9 system to genome editing in Leishmania, for future proof of concept and gene function studies, identification of new drug targets and possibly creating vaccinations by generating attenuated Leishmania strains.

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