Scientists at Harvard University have developed new CRISPR technology that allows the deletion of both copies of genes in diploid fungal strains.

Not without reason, significant attention has recently been focused on the need to develop novel anti-infective medicines that are effective against antibiotic-resistant bacteria. It is not only bacteria that are becoming increasingly resistant to existing therapies. Strains of the common fungal pathogen Candida albicans, which causes thrush and serious systemic infections, as well as forms biofilms on medical devices, are also becoming resistant to the currently available drugs used to treat them. 

C. albicans fungi are present on human skin and in the stomach, but can, particularly in people with weakened immune systems, become aggressive pathogens, leading to serious infections and sometimes death. Fighting the drug resistance of this fungus is challenging because each microbe is a “diploid” organism that contains two copies of its entire genome and of all the genes encoded within the genome. Identifying the link between a specific gene and its function requires the elimination of both copies of the gene at the same time, which has been difficult to do. The scientists are also challenged because some of the genes play similar or redundant roles in different processes, including drug resistance and biofilm formation. As a result, both copies of multiple genes need to be deleted at once to identify all of the genes involved.

To overcome this problem, researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University led by Core Faculty members James Collins and George Church, have developed a CRISPR-Cas9-based “gene drive” platform to create diploid strains of the pathogen in which both gene copies can be efficiently deleted.

The technology was developed using a rare “haploid” form of C. albicans that has only one set of chromosomes with one copy of each gene. This form of the fungus can be mated to create the diploid form. The scientists replaced genes of interest in the haploid form using a previously developed ‘gene drive’ that was adjusted to the specific biology of C. albicans. The modified haploids were then mated to form diploid fungi in which the ‘selfish genetic elements’ replaced the normal copy of the gene, according to Church, a Professor of Genetics at Harvard Medical School and of Health Sciences and Technology at Harvard and MIT. “The approach worked so efficiently that it enabled us to even delete pairs of different genes simultaneously with higher throughput and to explore whether their functions are related.”

The gene drive approach is based on the CRISPR-Cas9 system, in which a DNA-cutting Cas9 enzyme is targeted to two regions that flank a gene in haploid C. albicans fungi by two so-called guide RNAs (gRNAs). Once the targeted gene sequence has been removed, an engineered gene drive cassette expressing all Cas9 and gRNA components is inserted in its place. When two haploid fungi are mated to form diploid offspring, the gene drive substitutes the gene’s counterpart in the other chromosome, effectively deleting the original version from the organism.

Using this technology, the researchers identified combinations of genes that act synergistically to afford drug resistance or result in biofilm formation. “We can now get a much better handle on how genetic networks that underlie the virulence of C. albicans are organized, see how they respond to specific environmental and drug perturbations, and thereby uncover new vulnerabilities, that in the future may lead to new drug targets and combination therapies,” said Collins, who is the Termeer Professor of Medical Engineering & Science at Massachusetts Institute of Technology (MIT) and a Professor of Biological Engineering at MIT. “Moreover, our gene drive array platform can be a blueprint for similar approaches in other fungal pathogens, such as the newly emerging Candida auris, which is highly drug resistant and has already been marked as a threat by the Centers for Disease Control and Prevention.”