Date

2019 12:00 AM

Major

Biology

Department

Ecology, Evolutionary, and Organismal Biology

College

Agriculture and Life Sciences

Project Advisor

Josh Beck

Description

To continue fighting malaria, identification of new drug targets or life cycle intervention points and a better understanding of malaria parasite biology are crucial. While the ability to genetically manipulate the parasite in order to assess gene function is key to this goal, the tools to study important parasite genes are limited. CRISPR interference (CRISPRi) has emerged as a powerful and simple approach for conditional gene knockdown using enzymatically inactive Cas9 (dCas9). This project aims to develop and optimize a CRISPRi system in the most virulent human malaria parasite, Plasmodium falciparum, by determining optimal guide RNA design to achieve maximum target gene knockdown. To this end, we established a dual luciferase reporter system to test the activity of a tiled array of guide RNAs around the start codon of a gene target. We hypothesize that as the guide RNA location approaches the start codon, knockdown via dCas9 interference with transcription by RNA polymerase will increase. After determining the optimal guide RNA placement, the system can be used to screen putative transcriptional effector proteins to enhance knockdown. This system has the potential to provide a robust approach for studying essential P. falciparum genes with increased throughput. Current results will be discussed.

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Jan 1st, 12:00 AM

Optimizing CRISPR interference for Conditional Gene Regulation in malaria Parasites

To continue fighting malaria, identification of new drug targets or life cycle intervention points and a better understanding of malaria parasite biology are crucial. While the ability to genetically manipulate the parasite in order to assess gene function is key to this goal, the tools to study important parasite genes are limited. CRISPR interference (CRISPRi) has emerged as a powerful and simple approach for conditional gene knockdown using enzymatically inactive Cas9 (dCas9). This project aims to develop and optimize a CRISPRi system in the most virulent human malaria parasite, Plasmodium falciparum, by determining optimal guide RNA design to achieve maximum target gene knockdown. To this end, we established a dual luciferase reporter system to test the activity of a tiled array of guide RNAs around the start codon of a gene target. We hypothesize that as the guide RNA location approaches the start codon, knockdown via dCas9 interference with transcription by RNA polymerase will increase. After determining the optimal guide RNA placement, the system can be used to screen putative transcriptional effector proteins to enhance knockdown. This system has the potential to provide a robust approach for studying essential P. falciparum genes with increased throughput. Current results will be discussed.