New CRISPR Method Identifies Key Genes for Toxoplasma Survival

Using a new genetic screening approach with CRISPR, Crick researchers have identified key genes for parasite survival. Toxoplasma gondii In the mouse.

Their new method, published in Nature Communications, provides a new flexible approach that could expand the use of CRISPR screening. This method also makes it possible to simultaneously test hundreds of parasite genes in mice, which greatly reduces the number of search animals used.

Versatile library generation for CRISPR filtering

CRISPR screening is an extremely powerful way of identifying gene function. With the help of guide RNAs (known as gRNAs), specific genes are targeted and "inactivated". This elimination helps to determine which genes are essential for cell survival. However, these screenings are often done at the genome level and this scale can limit the types of experiments that can be done.

To overcome the limitations of genome-wide screening, the team has developed a new method for quickly generating and analyzing customized CRISPR banks of various sizes and compositions using a variety of methods. cloning in one step. This research was conducted by the signaling laboratory in Apicomplexan Parasites Lab, in collaboration with members of bioinformatics and high throughput screening facilities.

"This method of creating and analyzing gRNA pools targeting a subset of genes allowed us to perform CRISPR screenings much more quickly and transfer them into the in vivo infection model." ", explains the head of the Crick group, Moritz Treeck, lead author of the document.

"The RNA genes targeting the genes of interest are selected from a template and cloned and assembled into pooled CRISPR libraries.This custom CRISPR library is then transfected into the parasite to generate a pool of mutants that is then cultured at home. In mouse or cell culture, by sequencing the mutant pools of entry and exit, we are able to identify genes important for growth in any selection condition. "

New genes

Toxoplasma gondii is a parasite that affects about one-third of the human population. Although in a healthy individual, the symptoms associated with infection are few, the parasite poses a serious threat to AIDS patients. The development of the child in the uterus and the virulent strains responsible for eye diseases are widespread in South America. Previously, genome-wide genomic screening was used in vitro to identify the genes required for growth in cell culture, but screening methods to identify important genes for regulating the immune response of the genome. Host and ensure its survival in vivo, called "virulence factors". , "have not been available.

"By selecting a subset of genes to target, we did not take the risk of overloading the host before the parasite could spread," says Joanna Young, postdoc at Crick and co-author main document. "We had predicted that mice contaminated with Toxoplasma mutant pools would play a role in the interaction with the host, which revealed new virulence factors and confirmed those already known."

Interestingly, the team also found that some strains, unable to survive alone, were successful when co-infected in the mixed pool.

"For example, the MYR1 gene is required for the functions of many ToxoplasmaJoanna explains, "We were surprised to find that MYR1 inhibitors were still present and able to survive in mice when they were infected with other mutants. Now we need to know how it goes! "

Next steps

The next step is already underway. The team will collaborate with the Advanced Sequencing Facility to identify individual CRISPR mutants and complete RNA from the infected host cell to identify virulence factors that specifically interfere with the host response.