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.

Keywords: Plasmodium falciparum; CRISPR; biochemical techniques and resources; genetics; malaria.

Figure 1.. Overview of genetic screens in malaria parasites.

(A) Pools of mutants have been generated either through random mutagenesis using N-ethyl-N-nitrosourea (ENU), [23] or the piggyBac transposon system [24], (forward genetics) or with long homology arm gene targeting vectors [25], (reverse genetics). (B) Pools of mutants were selected for and propagated. (C) Mutant phenotypes were analysed through growth assays or other phenotypic assays such as microscopy or fluorescence activated cell sorting (FACS), where next-generation sequencing based methods were used to identify and or quantify the mutants. Quantitative insertion sequencing (QIseq), barcode sequencing (BarSeq). Here forward genetics refers to screens where genetic targets are not predetermined and reverse genetics where genetic targets are predetermined by the use of gene targeting vectors.

Figure 2.. The impact of genetic screens in malaria parasites.

Timeline of number of genes with phenotypes reported in (A) P. berghei (Pb) using data from the rodent malaria genetically modified parasite database [RgMDb, release 2022]. The inflection point from the Bushell et al. (2017), [25] genome scale screen is indicated. (B) P. falciparum (Pf) using data from PhenoPlasm [Phenoplasm, release 2022]. The inflection point from the Zhang et al. [24] genome scale forward genetic screen is indicated. (C) P. falciparum split by the genetic approach used, and excluding insertional mutagenesis (i.e. the Zhang et al. screen), showing a recent rise in the use of conditional knockout and knockdown approaches, and with the inflection point from the Maier et al. [6] knockout study indicated. Conditional knockout refers to deleting the gene at a specific time point/stage using dimerisable Cre-recombinase (DiCre). Knockdown here refers to a method where expression is inhibited at either the transcriptional, post-transcriptional, translational level, or the protein is inactivated or mislocalised. Natural deletion refers to spontaneous gene loss in e.g. in vitro culture.

Figure 3.. Overview of CRISPR/Cas9 Screens in Toxoplasma gondii.

Common to all CRISPR/Cas9 screening strategies is the construction of gene specific gRNA vector libraries. Cas9 can be expressed off the same vector as the gRNA or off a separate, co-transfected vector. Alternatively, the Cas9 enzyme can be integrated into the genome. For systems relying on the repair of Cas9-induced DSBs by homologous recombination the HDR template has to be supplied. Top panel: CRISPR–Cas9 knockout screen in T. gondii where (A) gRNA pools were prepared and (B) transfected into Cas9 expressing parasites that produce a decoy gRNA to minimise Cas9 toxicity. (C) gRNA sequencing was performed to identify genes important for parasite fitness in vitro [49]. Bottom panel: CRISPR/Cas9 knockdown screen in T. gondii where CRISPR/Cas9 was combined with the Auxin inducible degron (AID) system. (A) Pools of vectors carrying the gRNA and a repair template that introduce a mNeongreen (mNG)-AID tag were co-transfected with a Cas9 expressing plasmid. (B) Vector pools were transfected into a TIR1/IMC1 td-tomato line and allowed to propagate before arrayed and (C) pooled phenotypic analysis. The AID-tag targets the tagged protein for degradation by exogenous expression of transport inhibitor response 1 protein (TIR1). Inner membrane complex 1 (imc1) td-tomato expression was used in phenotypic assays [55].