Mutant/mutation
The mutant expresses a C-terminal 3xHA tagged version of CRTL

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2024.06.02.597011

Protein (function)
The gene/protein was identified in a 'homing screen' (see below for an explanation).
An AlphaFold 2 model predicts that PbCRTL has the drug-metabolite-transporter (DMT) fold, which in vertebrates is principally utilized by transporters for nucleotide-sugars and nucleotides. In lower organisms, however, bacterial proteins have been shown to serve as exporters of amino acids and their derivatives. Using FoldSeek, the closest experimental structure to PBANKA_0916000 is the Chloroquine Resistance Transporter from Plasmodium falciparum (PfCRT). OrthoDB identifies CRTL homologs in all Plasmodium species, but not in other Apicomplexa.

Phenotype
No expression in blood stages. Expression in ookinetes.
A time course of oocyst development detected CRTL-HA from day 2, where the label colocalised with highly refractile hemozoin crystals. Hemozoin in the oocyst is thought to originate from the female gametocyte which, like all erythrocytic stages, detoxifies heme moieties from hemoglobin digestion by aggregating them inside the digestive vacuole in crystalline form. Hemozoin is carried over into the oocyst where it apparently continues to reside inside the remnants of a DV-like compartment labelled by CRTL-HA. HA-positive granules are spread throughout the cytoplasm on days 2 and 4 of oocyst development. From day 6 they form aggregates, which do not increase in size as the oocyst grows. Around days 8-10 these labelled structures become forced into the narrowing spaces between growing sporogonic islands, where they continue to colocalise with the refractile pigment crystals, whose arrangement into lines or crosses is a hallmark of the oocyst at this stage. When oocysts sporulate, neither HA-tagged protein nor hemozoin crystals get incorporated into sporozoites. These results are consistent with the tagged protein residing in the DV remnants surrounding hemozoin crystals.

Analysis of a mutant lacking expression of CRTLRMgm-5580) showed the following:
No difference in the number of oocysts between wild type (WT) and crtl KO mutant on day 10 or 14. However, mutant oocysts were smaller on both days. Mutant oocysts stopped growing from day 8. By day 18, WT oocysts had grown to more than twice the diameter of mutant oocysts.
Crtl KO mutant oocysts lacked signs of sporulation. Mutant parasites also failed to produce salivary gland sporozoites compared to wild type on day 20. Mosquitoes infected with the mutant were unable to infect mice by bite, showing the gene is essential for transmission.

Additional information
From the paper:

Abstract:
In this study, we developed a scalable genetic system that uses barcoded gene targeting vectors equipped with a CRISPR-mediated homing mechanism to generate homozygous loss-of-function mutants to reveal gene functions in the functionally diploid life cycle stages. In this system, a knockout vector additionally expressing a gRNA for its target is integrated into one of the parental alleles and directs Cas9 to the intact allele after fertilisation, leading to its disruption. We find that this homing strategy is 90% effective in the oocyst, resulting in the generation of homozygous genotypes.

In the homing screen (using crosses between gametocytes of two lines making only male or only female gametocytes (fd1; RMgm-5578 and md4; RMgm-5579) it was found that two of three oocyst-expressed genes of unknown function, PBANKA_0916000 and PBANKA_1006400 showed a marked homing effect warranting further investigation. How mutants behave in the homing screen may be confounded by factors other than gene function, such as the efficiency of gRNAs, how much transcript or protein a zygote inherits from the female parent and how fast these turn over. To validate the result of the pilot screen we studied PBANKA_0916000 (RMgm-5580), a poorly understood gene with a new phenotype, which we find encodes a chloroquine resistance transporter like (CRTL) protein.

Single sex P. berghei lines expressing Cas9-BFP are fertile
Sex in haploid malaria parasites is not chromosomally determined, and parasite clones produce both male and female gametocytes. We reasoned that if we could genetically modify lines to produce gametes of only one or the other sex, these could be engineered further, such that in a genetic cross each sex would separately deliver either a Cas9 endonuclease or a guide RNA to the zygote and cause a double strand break in a target gene. Since Plasmodium parasites lack canonical non-homologous end joining, repair of double strand breaks is always homology driven. We therefore further reasoned that a disrupted target allele not recognized by the guide and carried by one of the parental lines would serve as repair template, leading to the disruption being copied from one parental genome to the other, generating a homozygous knock-out (KO).
To test this concept, we first created lines making only male or only female gametocytes by disrupting female development 1 (fd1; RMgm-5578) or male development 4 (md4; RMgm-5579), respectively. Clonal md4-cas9-bfp (female-only) parasites produced female gametes and did not produce male gametes and gave rise to ookinetes only when co-cultured with the fd1-cas9-bfp (male-only), which did release male gametes, as expected.  Also as expected, these single-sex lines produced no oocysts when transmitted individually, but oocyst formation was restored when mosquitoes were fed on mice co-infected with both lines.

Cas9-mediated genome editing after fertilisation is efficient.
To quantitate the efficiency of Cas9-mediated genome editing after fertilisation at the level of individual oocysts, we designed a colour swapping experiment. This involved constructing complementary single-sex lines in which the endogenous MyoA protein was either fused to mCherry or to GFP. MyoA is abundantly expressed during sporogony and tolerates c-terminal tagging12. We hypothesised that crossing single-sex lines expressing MyoAmCherry and MyoA-GFP, respectively, should make yellow oocysts. However, if the latter locus additionally expressed one or more gRNAs targeting Cas9 specifically to the mCherry locus , homology-mediated repair of a double-strand break would use the complementary GFP-containing allele as repair template, turning oocysts green.
Consistent with this concept, directional crosses between red and green parents produced yellow oocysts, unless either the female or the male gamete provided gRNAs targeting the mCherry locus (all crosses also delivered Cas9 to the zygote). Since md4 and fd1 mutants do not undergo selfing, we were surprised to see 1-3% of oocysts in these crosses express only one parental allele, indicating that some zygotes may obtain an incomplete genome from a parent or that occasionally not all four products of meiosis survive or replicate in the oocyst. Our data suggest homing happens with 97% efficiency when the gRNAs are expressed from the female gamete, but this is reduced to 89% if the male carries the guides. This colour swapping experiment demonstrates that we have developed a CRISPR method where the phenotype of the oocyst is dominated by only one parental allele, in this case myoa-gfp, irrespective of the route of inheritance.

Other mutants