In a new study, researchers from the Francis-Crick Institute in the United Kingdom identified key genes for the survival of Toxoplasma gondii in mice by using a CRISPR-based gene screening method. Related research results were recently published in the journal of Nature Communications. This study provides a flexible new approach to extending the use of CRISPR screening, which also allows for the simultaneous testing of hundreds of genes in Toxoplasma in mice, thereby greatly reducing the number of research animals used.
CRISPR screening is an extremely effective method for identifying gene function. Specific genes are targeted for "knockout" by using a guide RNA (gRNA). This knockout helps determine which genes are critical for cell survival. However, these screens are usually performed across the genome, and the size limits the types of experiments scientists can process.
To overcome the limitations of whole-genome screening, these researchers have developed a new approach to rapidly generate and analyze custom CRISPR libraries of different sizes and compositions using a single-step cloning approach. Moritz Treeck, author of the paper, said that, "This method of constructing and analyzing gRNA libraries that target only a subset of genes allows us to perform CRISPR screenings faster and transfer them to in vivo infection models. "
The gRNAs that target the gene of interest are selected from a set of sequences that are subsequently cloned and assembled into a pooled CRISPR library. This custom CRISPR library is then transfected into Toxoplasma gondii to generate a library of mutants, which are then cultured in mouse or cell culture. By sequencing the input and output mutant libraries, researchers were able to identify genes that are important for growth under any chosen condition.
Toxoplasma gondii is a parasite that affects approximately one-third of the population. Although there are few infection-related symptoms in healthy individuals, Toxoplasma gondii is a serious threat to AIDS patients and children in the uterus, and pathogenic Toxoplasma gondii strains causing eye diseases are ubiquitous in South America. Prior to this, genome-wide gene screening has been used in vitro to identify genes required for growth in cells cultured in vitro, However, there are currently no screening methods to identify genes known as "virulence factors" that are critical for regulating host immune responses and ensuring survival in vivo.
Joanna Young, a co-first author and postdoctoral fellow at the Francis-Crick Institute, said, "By targeting a subset of genes for targeting, we do not risk the overwhelming host before Toxoplasma can reproduce. We infected mice with a Toxoplasma mutant library and predicted that they would interact with the host. This reveals new virulence factors and confirms those virulence factors known in previous studies."
Interestingly, these researchers also found that some Toxoplasma strains that were unable to survive on their own were able to survive successfully in mice infected with mixed mutant libraries. Joanna explained, "For example, the MYR1 gene is required for many Toxoplasma secretion proteins to function. Without it, the parasite could not initiate infection. However, we were surprised to find that when infected with other mutants, mice were infected. At the time, the MYR1 knockout mutant was still able to survive in mice. We need to find out how this happens."
The next phase of research is already underway. These researchers wanted to identify intact RNAs from individual CRISPR mutants and infected host cells to identify virulence factors that specifically interfere with host responses.
Reference
- Joanna Young et al. A CRISPR platform for targeted in vivo screens identifies Toxoplasma gondii virulence factors in mice. Nature Communications, 2019