Whole‐genome CRISPR screens to understand Apicomplexan–host interactions

Apicomplexan parasites are aetiological agents of numerous diseases in humans and livestock. Functional genomics studies in these parasites enable the identification of biological mechanisms and protein functions that can be targeted for therapeutic intervention. Recent improvements in forward genetics and whole‐genome screens utilising CRISPR/Cas technology have revolutionised the functional analysis of genes during Apicomplexan infection of host cells. Here, we highlight key discoveries from CRISPR/Cas9 screens in Apicomplexa or their infected host cells and discuss remaining challenges to maximise this technology that may help answer fundamental questions about parasite–host interactions.

to generate a double-strand break at a predetermined site and swiftly generate gene-depleted parasites at an unprecedented pace (Straimer et al., 2012).
Briefly, after inducing DNA or RNA double-strand breaks (DSBs) using an RNA-guided (sgRNA) endonuclease, the repair of these breaks can follow two main pathways: non-homologous end joining (NHEJ) and homologous recombination (HR), as well as microhomology-mediated end joining (MMEJ) (Sfeir & Symington, 2015).NHEJ often results in small insertions or deletions (indels) at the break site, potentially leading to functional gene knockout due to frameshift mutations.However, if NHEJ is deficient or inhibited, HR or MMEJ can come into play.(Micro) homology recombination relies on (short) homologous sequences near the DNA break site for repair, and it's notable for its ability to utilise these homologies to align and repair the broken DNA ends.
When a homologous template is provided along with the sgRNA, it allows for HR repair, which is particularly useful in NHEJ-deficient cells.
Nonetheless, CRISPR/Cas9 significantly improved the efficiency of genome editing by directing DSB at target loci in multiple parasitic cells concurrently.In P. falciparum for example, this reduced the time required to generate clonal transgenic parasites with genomic integration from 2 to 12+ months using traditional molecular approaches to less than 1 month (Ghorbal et al., 2014;Maier et al., 2008).
In addition to using CRISPR/Cas9 for reverse genetics, the use of this technology in forward genetics studies has greatly improved the understanding of parasite-host interactions, stage differentiation, virulence, drug resistance and pathogen-host interactions in Apicomplexa.This review discusses how CRISPR/Cas9 genetic screens have improved the understanding of parasite-host interactions, discusses CRISPR/Cas challenges to be overcome for its use in Apicomplexa and how this screening technology can be further employed in the future.

| FORWARD G ENE TI C SCREENING IN APICOMPLE X A : B EFORE CRIS PR /C A S
Prior to CRISPR/Cas technology, common forward genetic techniques included genetic crosses, quantitative trait locus (QTL) mapping, genome-wide association studies, N-ethyl-Nnitrosourea mutagenesis, microarray profiling, overexpression, insertional mutagenesis via viruses or transposons, RNA interference (RNAi) screens and drug selection screens.For example, QTL mapping identified virulence factors in Toxoplasma and the genetic basis for macrophage responses to Toxoplasma infection (Hassan et al., 2015;Saeij et al., 2006;Taylor et al., 2006).In P. falciparum, QTL mapping aided the identification of the chloroquine resistance transporter gene, crt (Wellems et al., 1990) and reticulocyte binding protein homologue polymorphisms (Campino et al., 2018;Hayton et al., 2008).Comparative microarray profiling discovered essential genes for gametocytogenesis in P. berghei (ApiAP2 family) (Sinha et al., 2014).PiggyBac transposon-based mutagenesis uncovered the essential genome of different malaria parasites, leading to a deeper understanding of parasite blood stage biology (Balu et al., 2009;Zhang et al., 2018).Examples of drug selection screens include the identification of molecular markers of P. falciparum artemisinin resistance (Ariey et al., 2014;Tucker et al., 2012).
Of these forward genetic techniques, RNAi is most similar to CRISPR gene editing.Many Apicomplexa lack or have noncanonical RNAi machinery, limiting its use in Apicomplexan research.
However, RNAi screening in host cells identified regulators of actin dynamics as important in Toxoplasma invasion (Gaji et al., 2013), unveiled host genes involved in Plasmodium invasion of erythroblasts (Egan et al., 2015) and hepatocytes (Prudêncio et al., 2008), and hepatocyte defence mechanisms (Raphemot et al., 2019).RNAi screens have also been important in identifying potential targets for drug and vaccine development.However, RNAi has limitations: small interfering RNAs can be degraded, can enter target cells inefficiently, and its knockdown effects are short-lived requiring repeated dosing.

| CRIS PR /C A S KNO CK-OUT SCREEN S: A P OWERFUL APPROACH FOR FORWARD G ENE TIC S TUD IE S IN API COMPLE X AN PA R A S ITE S
CRISPR/Cas comprises useful methods for conducting forward genetic screens that provide advantages over traditional gene editing approaches.They can target DNA or RNA such that gene expression is disrupted via frameshift or insertion (CRISPRko), transcriptional interference (CRISPRi/a) or individual nucleotide changes (base and prime editing).CRISPR/Cas screens can be conducted in pooled or arrayed formats (Figure 1).In pooled CRISPRko screens, sgRNA libraries are introduced into Cas-expressing target cells, resulting in a heterogeneous pool of genetic perturbations and the identification of hit gene candidates through selection pressure and comparison with control pools.
Cas9 is the only enzyme so far used in pooled forward genetic screens with Apicomplexa.Such screens involved Toxoplasma since they possess NHEJ.Stable expression of Cas9 in Toxoplasma can be toxic, though this was elegantly overcome using a decoy sgRNA to minimise unintended Cas9 activity (Sidik et al., 2016).This enabled the first genome-wide CRISPR/Cas9 loss-of-function screen in Toxoplasma, identifying fitness-conferring genes critical for invasion of the host cell, including clamp (claudin-like Apicomplexan microneme protein) (Table 1).Phenotypic validation studies confirmed CLAMP's importance in Toxoplasma invasion and the P. falciparum lytic cycle as well as in sporozoites (Loubens et al., 2023).
Whole-genome CRISPR/Cas9 knockout screens also identified parasite genes associated with drug resistance, and susceptibility (Harding et al., 2020;Sidik et al., 2016).For example, a screen in Toxoplasma revealed a porphyrin transporter homologue, tmem14c, whose genetic disruption increased susceptibility to dihydroartemisinin, whilst disruption of the mitochondrial protease degp2 increased resistance to the drug in both Toxoplasma and P. falciparum.(Harding et al., 2020).These findings demonstrate the power of model organisms like Toxoplasma for conducting comprehensive whole-genome studies that are of direct relevance to other Apicomplexa.
Other CRISPR/Cas9 knockout screens in Toxoplasma identified genes (gra45, facilitates the secretion of Toxoplasma proteins, extending beyond the PMV (Wang et al., 2020); six genes of which five encode GRAs (Krishnamurthy et al., 2023); gra57, gra70 and gra71 which encode proteins that form a complex that boost parasites' capacity to persist in IFNγ-activated fibroblasts (Lockyer et al., 2023)) that regulate parasite growth in naïve and IFNγ-activated macrophages or IFNγ-activated fibroblasts, respectively.A CRISPR/ Cas9 knockout screen of ~200 Toxoplasma genes was also used to study the transition from tachyzoites to latent bradyzoites, a critical step in chronic infections.This approach identified differentiation regulators like the Myb-like transcription factor BFD1 (Bradyzoite-Formation Deficient 1), which was validated to be essential for developing chronic parasite stages in vitro and in vivo (Table 1) (Waldman et al., 2020).Furthermore, comparing fitness-conferring genes in screens with WT and specific knock-out parasites can reveal protein connections.Paredes-Santos et al. ( 2023), for example, found that GRA72 localised with GRA17/GRA23, by screening WT and Δgra17 parasites.
In vivo Apicomplexan CRISPR/Cas knockout screens can provide a deeper understanding of infection biology in both the parasite and the host (Sangare et al., 2019;Young et al., 2019).For example, in vitro-generated Toxoplasma knock-out libraries followed by in vivo selection pressure experiments in mice established the importance of specific genes for colonisation of distant organs and parasite virulence in animal hosts.For example, the gene encoding TgWIP (Toxoplasma WAVE-complex-interacting protein) was found to be essential for Toxoplasma interactions with the host's WAVE regulatory complex, increasing motility and dissemination of infected cells throughout the host (Sangare et al., 2019).Furthermore, through CRISPR knockout screening, ROP1, the initial rhoptry protein identified in Toxoplasma, was identified to play a crucial role in virulence and previously unrecognised resistance to interferon gamma-mediated innate immune restriction (Butterworth et al., 2022).This demonstrates the potential of in vivo CRISPR screens to also uncover previously unknown host cell interaction partners, in addition to Apicomplexan interaction partners, that are linked with parasite fitness and spread.
A limitation of screening approaches is that many genes are essential for growth and therefore may not be directly represented in the screen or assigned a specific function.A conditional CRISPR/Cas knockout screen was recently developed to overcome these shortcomings.A library of conditional gene knock-out Toxoplasma parasites was screened for regulators of actin dynamics during the lytic stage of growth in host cells (Li et al., 2022).Rapamycin-inducible, dimerisable split Cas9 (sCas9) was employed along with parasites expressing an Factin chromobody and red fluorescent protein to track parasite-host F I G U R E 1 CRISPR/Cas screen workflow.CRISPR/Cas screens are classified as pooled (heterogenic bulk population of mutants) or arrayed (separate gene knockouts) screens.The sgRNA library is integrated into Cas-expressing target cells to produce target cells with genetic perturbations.Selection pressure is applied to identify resistant or sensitive candidate cells, and sgRNA abundance is compared to a control pool to identify hit gene candidates.Validation of hits is typically required.interactions during infection.Toxoplasma in which cgp (conoid gliding protein) or slf (signalling linker factor) were disrupted resulted in parasite egress phenotypes such that CGP was required for gliding motility and SLF was involved in phosphatidic acid signalling required to initiate egress.This study showcases the potential of CRISPRmediated knockout screening by combining conditional Cas9 systems with genetically modified parasites to elucidate novel biology.

| VER SATILIT Y OF CRIS PR /C A S: APICOMPLE X AN SCREEN S VIA REG UL ATED G ENE E XPRE SS I ON
In contrast to knockout screens, pooled CRISPR-mediated gene interference and activation (CRISPRi/a) screens enable precise and targeted control of gene expression without disrupting genes.This technology uses catalytically impaired nucleases like dead Cas9 (dCas9) or Cas9 nickase (Cas9n) and offers versatile avenues for genetic modulation.When associated with gene repression or transcriptional activator domains, these enzymes enable control of gene expression.Additionally, their integration with single-stranded DNA-specific nucleobase modification enzymes, known as base editors, further expands the toolkit for precise and targeted genetic modifications, however, this innovative technology has yet to find application in Apicomplexan research (Figure 2).CRISPRi/a has been exploited in Apicomplexa that lack canonical NHEJ activity, such as Plasmodium and Cryptosporidium (Baumgarten et al., 2019;Walker & Lindner, 2019;Xiao et al., 2019), allowing the study of essential proteins in specific life stages.This however has only been done in arrayed screening formats which is much more labour intensive compared to pooled screen and highlights opportunities for potential use in Apicomplexa (Figures 1 and 2).Combining CRISPR/Cas technology with HR facilitates the integration tags, fluorophores or degrons, enabling the study of gene function and the induction of conditional gene knockouts with precision and control.The first CRISPR/Cas-mediated HR pooled knockdown screen in Toxoplasma that employed CRISPR-mediated interference investigated over 100 modified kinases using a high throughput tagging strategy (Table 1) (Smith et al., 2022).By fusing an auxin-inducible degron to mNeonGreen, CRISPR-directed HR was used to endogenously label kinases and study their function in different parasite life stages.This approach allowed high throughput tagging with optional protein downregulation through the addition of auxin indole-3-acetic acid (Smith et al., 2022).The novel kinase SPARK (store potentiating/activating regulatory kinase) was identified to play a role in regulating Ca 2+ release during parasite egress and invasion.
In summary, pooled CRISPR/Cas screening has been successfully conducted in Toxoplasma using Cas9 due to the presence of NHEJ in this parasite.These screens have augmented the understanding of Toxoplasma infection biology and the broader field of Apicomplexan biology.Moreover, since many parasites lack NHEJ, host-directed screens present a promising alternative to pooled forward genetics screening, leveraging the well-established CRISPR/Cas toolbox for mammalian cells for parasitic infection.Host-directed screens offer a powerful approach to uncover the interplay between host and pathogen, revealing insights into host factors, pathogenic mechanisms, and potential therapeutic targets which are further discussed below.

| P OOLED CRIS PR /C A S K NO CKOUT SCREEN S OF THE APICOMPLE X AN -INFEC TED HOS T CELL
Existing CRISPR systems in murine and human cells, along with prevailing sgRNA libraries and vector delivery systems, provide a promising platform for studying host factors in Apicomplexan infection.Host-directed screens involve perturbing host gene expression using the CRISPR/Cas system and observing the effects on parasitic infection and survival (Table 2).In the context of Toxoplasma, the first CRISPR/Cas9 screen of host genes revealed over 1000 host dependency factors essential for infection, with 26 host genes associated with inhibiting parasite growth during type I interferon signalling (Gossner et al., 2022;Wu et al., 2019).
Enrichment analysis agreed with previous RNAi screens (Gaji et al., 2013) and highlighted the importance of cell-cell adhesion, cell cycle regulation, protein heteromerization, IL1β production and transcription factor SUMOylation for Toxoplasma growth and host cell survival.
Cryptosporidium parvum is an important Apicomplexan parasite that causes severe symptoms in immunocompromised patients and is acquired through ingestion of contaminated food or water.
A host-directed CRISPR/Cas9 knockout screen investigated host genes that influence C. parvum infection and host cell survival, covering ~19,000 human protein-coding genes (Gibson et al., 2022).
The screen revealed that the host interferon response type III via  (Gossner et al., 2022) In summary, host-directed CRISPR/Cas9 knock-out screens have revealed novel host factors and pathways involved in infection by Toxoplasma, Cryptosporidium and Plasmodium spp.However, there are still opportunities to leverage and knowledge to learn regarding the complex interaction of parasites with their host cells.The use of alternative Cas enzymes, such as Cas13 or dCas, may be beneficial for studying Apicomplexan-host interactions for example by performing CRISPRi/a screens of host factors.The CRISPR toolbox is constantly expanding and should continue to deepen our understanding of Apicomplexa-host interactions, yet its full capacity remains untapped.

| CON CLUS I ON S AND FUTURE DIREC TIONS
Medium and high throughput CRISPR/Cas genome screens have been successfully conducted in Apicomplexa and their host cells, primarily using Cas9, to elucidate important new knowledge.While Cas9 shows continued promise for future parasite research, opportunties remain for broadening the utility of CRIPSR/Cas for efficient and precise genetic manipulation of Apicomplexa.
CRISPR/Cas systems are functionally diverse and modular.Cas enzymes such as Cas12a, Cas3, Cas13, Cas14, Cas7-11 offer different features in terms of their size, structure, and nucleic acid target specificity (reviewed in Hillary & Ceasar, 2022) that may be useful in functional parasitology studies.Cas12a, which is smaller than Cas9, has demonstrated high efficiency in targeting spacer sequences that follow a 5′T-rich protospacer adjacent motif (PAM) sequence, enabling targeting of different regions in the genome.This could be advantageous for Plasmodium spp.due to the high ratio of A/T nucleotides in their genomes.Cas12a also produces sticky DNA ends, which enhances HR-mediated repair.Alternatively, Cas3 degrades single-stranded DNA, enabling selective removal of long DNA sequences.However, its selective removal of large DNA sequences can sometimes be imprecise, as Cas3 often dissociates from the cascade during the cutting process (Huo et al., 2014) so this system requires careful validation in Apicomplexa.Cas14, a family of very small RNA-guided nucleases (400-700 aa), precisely cuts singlestranded DNA without specific PAM requirements.Moreover, target recognition activates its trans-cleavage property which allows for nucleotide polymorphism genotyping, also common with other type V Cas enzymes, such as Cas12a and b (Chen et al., 2021;Harrington et al., 2018), providing opportunities for diagnostics.Furthermore, Type VI Cas enzymes (e.g.Cas13) can cleave RNA and repress gene expression, whereas the RNA-targeting Type III Cas enzymes (e.g.Cas7-11 complex or Csm complex) are known to activate RNA transcription non-specifically (Staals et al., 2014).Such enzymes may provide useful for the future study of parasite infection by enhancing DNA recombination or allowing regulation of RNA.
While the expanding CRISPR/Cas toolbox is amenable to adaptation to Apicomplexan research to address various aspects of parasite development and host interaction, some CRISPR systems (e.g.Cas12a) might be more feasible for high-throughput screening than others.Interestingly, while Cas13 presents challenges due to its cellular toxicity and activation of the siRNA pathway, Plasmodium lacks robust RNA interference machinery.Consequently, it remains to be determined whether Cas13 is a suitable enzyme for high-throughput screening.Finally, exploring dCas endonucleases for activation or interference screens in Apicomplexa and their hosts offers promise for finely tuned genetic manipulation for functional studies.In conclusion, CRISPR screening has revolutionised the study of Apicomplexan infection biology and yet there are still many opportunities to pursue.Ongoing optimisation of CRISPR screens, utilisation of new Cas enzymes and sgRNA libraries and continued application to in vivo studies will further enhance our understanding of these fascinating and medically important parasites.

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I G U R E 2 Common CRISPR approaches for gene editing and whole genome screening include CRISPRko (knock-out), CRISPRi (interference), CRISPRa (activation), base editing and prime editing.CRISPRko involves Cas-induced double-strand breaks (DSB) repaired by NHEJ, resulting in gene knockouts.CRISPRi utilises Cas endonucleases fused to transcription repression domains to reduce target gene transcription.CRISPRa employs Cas endonucleases linked to transcription activation domains to increase target gene transcription.Base editing introduces point mutations without DSB using a base editor.Prime editing uses Cas endonucleases fused to a reverse transcriptase to insert or delete nucleotide sequences without DSB.interferon-lambda and toll-like receptor 3 (TLR3), played a protective role against C. parvum infection.Additionally, glycosaminoglycan synthesis and glycosylphosphatidylinositol anchors were crucial for parasite development and host cell viability during infection.This study successfully uncovered novel host pathways involved in C. parvum infection biology using CRISPR/Cas9 gene editing, RNA sequencing and protein analyses.CRISPR screens targeting Plasmodium in red blood cells is challenging due to the host cells lacking a nucleus and the limitations of their lifespan and machinery for genetic modification.An alternative approach is to modify early red blood cell progenitors or erythroblasts during ex vivo erythropoiesis, creating modified cells for arrayed screening.Importantly however, next-generation sequencing post-selection pressure remains unfeasible once the red blood cells lose their nuclei.So far, one screen using shRNA instead of CRISPR-Cas9 has been reported, identifying CD44 and CD55 as new factors influencing Plasmodium invasion (Egan et al., 2015).Genetic host screens targeting the Plasmodium liver stage offer important insights into the initial phase of malaria infection by sporozoites.They are particularly valuable as modifying hepatocytes is comparatively more feasible than altering red blood cells.The first genome-wide CRISPR/Cas9 host screen involving Plasmodium spp.identified hepatocyte factors that contribute to invasion and intracellular development by Plasmodium yoelii (Vijayan et al., 2022).The study highlighted the importance of microtubule-mediated vesicular transport and retrograde trafficking for parasite liver stage development.Knockout of the centrosomal protein CENPJ, which increases localisation of non-centrosomal microtubule organising centres to the parasite periphery, increased parasite replication during liver stage development.The low-density lipoprotein receptor-related protein 4 (LRP4) was also found to positively regulate P. yoelii invasion of hepatocytes.The authors demonstrated that current gene enrichment analyses have limitations in capturing complex host-parasite interactions, necessitating the development of novel analytic platforms for a comprehensive understanding of Plasmodium-hepatocyte pathways.Another CRISPR/Cas9 host knock-out screen targeted ~500 hepatocyte genes associated with pathogen entry receptors and investigated P. berghei sporozoite invasion(Lee, 2020).Host integrin subunit alpha V (itgav) and integrin subunit beta 5 (itgb5) were identified as hits, but validation experiments could not confirm if their function was specifically involved in sporozoite invasion.This study highlighted the complexities of establishing host cell-directed CRISPR/Cas9 screens for understanding Plasmodium sporozoite infection.Due to the complexity and technical challenges associated with studying P. falciparum liver stages, no CRISPR/Cas screen of hepatocytes has been published with human-infecting Plasmodium parasites to date, though this is a focus in our laboratory.It may be possible to employ this technology to unveil host factors involved in sporozoite cell traversal since the rates of this phenotype are significantly higher than for productive invasion of cultured hepatocytes including organoids.TA B L E 2 CRISPR/Cas screens for host factors during Apicomplexan infection.
CRISPR/Cas screening technology offers researchers the opportunity to explore unresolved future questions.For example, what are the factors and mechanisms involved in parasite dormancy?What further details can be elucidated on the regulators and effectors of stage differentiation, parasite transmission and host immune responses?In vivo CRISPR screens may answer questions around the importance of host micro-and macroenvironment associated with Apicomplexan infection.While such screens come with challenges such as targeted delivery and off-target effects, careful optimisation may lead to success commensurate with the effort.

TA B L E 1
Summary of pooled CRISPR/Cas screens in Apicomplexa.