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CRISPR Screen Reveals Genes Essential to Toxoplasmosis, Malaria Parasites

NEW YORK (GenomeWeb) – Genome-wide knockout screens to find essential genes are proving useful not only in cancer research, but also in parasitology. A new study published today in Cell describes a method to find essential genes in Toxoplasma gondii, which could also help scientists better understand the biology of the closely-related malaria parasite, Plasmodium falciparum.

Led by co-first authors Saima Sidik and Diego Huet and senior author Sebastian Lourido of the Whitehead Institute for Biomedical Research, a team of scientists from several institutions presented the first genome-wide screen of a member of the phylum Apicomplexa — one-celled parasitic protists that include malaria parasites and water-borne parasites in the genus Cryptosporidium.

"This is an important leap forward in what's possible to investigate in these parasites," Lourido said in a statement.

After tweaking the CRISPR/Cas9 system for maximum gene disruption in T. gondii, the researchers found approximately 200 previously uncharacterized fitness-conferring genes, out of more than 8,000 protein-coding genes. They determined that one such "indispensable conserved apicomplexan protein" (ICAP) is an invasion factor conserved throughout the phylum, and its P. falciparium ortholog is essential during the asexual cycle of that parasite. They also found genes involved in drug sensitivity.

The investigators framed the study as a window into malaria parasite biology, which has proved difficult to study with CRISPR/Cas9. "Because its genome is adenine- and thymine-rich, it is difficult to generate the cuts where you want," Huet said. Cas9 requires an NGG recognition site adjacent to a genomic target. CRISPR/Cpf1, which recognizes a TTN site, has been proposed as a tool for editing in malaria parasites. But the parasites also lack the indel-forming, non-homologous end-joining pathway of DNA repair. "These issues represent a technical hurdle to similar genome-wide approaches in this parasite, making Toxoplasma an even more important model for malaria," he added.

The US Centers for Disease Control and Prevention estimates that T. gondii infects more than 60 million people in the US, usually without symptoms, but can lead to toxoplasmosis, a mild illness. Like the malaria parasite, T. gondii proved somewhat resistant to CRISPR/Cas9 editing. Regular Cas9 activity proved to be toxic to the one-celled organism, so the scientists provided a dummy guide RNA to reduce editing activity.

Once optimized, the CRISPR/Cas9 screen consisted of 10 guides against each of 8,158 predicted protein-coding genes. Among the 200 or so ICAPs, the scientists identified one that seemed to be found only in Apicomplexa, suggesting it was important to the parasitic nature of those organisms. Bioinformatic analysis suggested a structural similarity to mammalian transmembrane proteins claudin-15 and -19, so the researchers dubbed it "claudin-like ampicomplexan microneme protein" (CLAMP). Further experiments showed that it is required during the invasion of human fibroblasts. The researchers wrote that they took video of CLAMP-less parasites "repeatedly pushing against the host cell membrane while failing to initiate invasion."

Moving to the malaria parasite, the scientists showed that CLAMP ortholog knockdowns blocked the organism's asexual cycle, although its precise function in that organism remains unknown.

"Coupled with the diverse tools available for genetic and chemical manipulation of T. gondii, the genome-wide screens will provide a framework for the systematic examination of genetic interactions," the authors wrote. "Future screens will need to define the genes required during other life stages, in different hosts, under varying nutrient conditions, and in response to immune pressures."

According to the authors, the study heralds a new, exciting phase in malaria biology research. "This technology can be used to study a variety of topics, from nutrient acquisition and responses to immune pressures to epistasis, and genetic interactions," Lourio said.

Sidik added that the study would help speed up research. "We can knock down the whole genome in a week, whereas before we could only do maybe one gene a month," she said. "With CRISPR screening, the possibilities are endless."