CRISPR/Cas9 and genetic screens in malaria parasites: small genomes, big impact

Abstract

The ∼30 Mb genomes of the Plasmodium parasites that cause malaria each encode ∼5000 genes, but the functions of the majority remain unknown. This is due to a paucity of functional annotation from sequence homology, which is compounded by low genetic tractability compared with many model organisms. In recent years technical breakthroughs have made forward and reverse genome-scale screens in Plasmodium possible. Furthermore, the adaptation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Associated protein 9 (CRISPR/Cas9) technology has dramatically improved gene editing efficiency at the single gene level. Here, we review the arrival of genetic screens in malaria parasites to analyse parasite gene function at a genome-scale and their impact on understanding parasite biology. CRISPR/Cas9 screens, which have revolutionised human and model organism research, have not yet been implemented in malaria parasites due to the need for more complex CRISPR/Cas9 gene targeting vector libraries. We therefore introduce the reader to CRISPR-based screens in the related apicomplexan Toxoplasma gondii and discuss how these approaches could be adapted to develop CRISPR/Cas9 based genome-scale genetic screens in malaria parasites. Moreover, since more than half of Plasmodium genes are required for normal asexual blood-stage reproduction, and cannot be targeted using knockout methods, we discuss how CRISPR/Cas9 could be used to scale up conditional gene knockdown approaches to systematically assign function to essential genes.

Toolkit of Approaches To Support Target-Focused Drug Discovery for Plasmodium falciparum Lysyl tRNA Synthetase

“…This is in part due to the success of phenotypic approaches but also partly enforced by the lack of robustly validated drug targets in Plasmodium. However, the emergence of improved genetic tools for the study of this apicomplexan parasite has enabled many more viable drug targets to be identified and robustly validated. ,, This, coupled with the fact that the massive screening efforts of organizations such as Medicines for Malaria Venture has explored and now exhausted much of the available chemical space, has led to a renewed focus on target-based drug discovery. To support this decided change of emphasis, the development of additional tools and methodologies will be required.…”

Gene-by-gene screen of the unknown proteins encoded onP. falciparumchromosome 3

“…However, they provide no data for the cellular location of individual proteins nor cell lines for their functional analysis. For instance, Zhang et al (2018) provides a predicted score of essentiality for P. falciparum genes that – despite its usefulness – does not preclude experimental validation for individual targets of interest (Ishizaki et al, 2022). These latter issues are solved by gene-by-gene screens that however typically lack through-put in malaria parasites (Ishizaki et al, 2022; de Koning-Ward et al, 2015; Rancati et al, 2018).…”

“…For instance, Zhang et al (2018) provides a predicted score of essentiality for P. falciparum genes that – despite its usefulness – does not preclude experimental validation for individual targets of interest (Ishizaki et al, 2022). These latter issues are solved by gene-by-gene screens that however typically lack through-put in malaria parasites (Ishizaki et al, 2022; de Koning-Ward et al, 2015; Rancati et al, 2018). Taking advantage of the selection-linked integration (SLI) system for genomic modification (Birnbaum et al, 2017) we here carried out a proof-of-concept study covering all unknown ‘non-secretory’ proteins (i.e.…”

Plasmodium yoelii surface-related antigen (PySRA) modulates the host pro-inflammatory responses via binding to CD68 on macrophage membrane

“…The proposed CRISPR/Cas9 gene editing technique was used to screen the PySRA knockout strain in vivo to further explore the specific regulation mechanism of PySRA on the host inflammatory response. The CRISPR/Cas9 technique comprises Cas9 endonuclease and guides RNA (gRNA), which helps Cas9 achieve specific recognition and cleavage of target sequence sites ( 37 – 39 ). In this study, no resistant strains were found after electroconversion and pyrimethamine screening.…”