New gene drive targeting a fungal pathogen enables identification of virulence regulators and potential future combination therapies.

Candida albicans is a notorious human fungal pathogen that causes thrush and serious systemic infections. Opportunistic C. albicans fungi, which often live inconspicuously in the normal flora of human skin and gut, can switch from their harmless stealth mode to become aggressive pathogens, especially in people whose immune systems are already compromised by pre-existing diseases or harsh drug therapies. They can also form biofilms on medical devices, such as catheters and stents in the human body, leading to infections and sometimes death. The threat posed by both free and biofilm-bound forms of the pathogen is constantly growing, as virulent C. albicans strains are becoming increasingly resistant to the few drugs that are available to treat them.

Microbiologists are facing tremendous difficulties in their quest to fight C. albicans’ drug resistance and biofilm formation. Each C. albicans microbe is a “diploid” organism, as it usually contains two copies of its entire genome and of all the genes encoded within. However, to understand the role that a specific gene plays, researchers need to be able to delete both copies at the same time, allowing them to observe the effects of the gene’s total absence, which has been a difficult challenge in C. albicans. In addition, genes often play very similar and sometimes redundant roles in many processes, including drug resistance and biofilm formation, meaning that more than one gene needs to be deleted to identify those genes whose functions are linked.

To approach the gene deletion challenge in C. albicans, a collaborative team led by James Collins and George Church, two Core Faculty members at Harvard’s Wyss Institute for Biologically Inspired Engineering, have developed a CRISPR-Cas9-based “gene drive” platform to create diploid strains of the pathogen in which both gene copies could be efficiently deleted. The technique may lead the way toward a better understanding of drug resistance and biofilm-forming mechanisms, and through future research, it could help pinpoint new drug targets and combination therapies.

The study was published in the journal Nature Microbiology.

The team took advantage of a recently discovered very rare “haploid” form of C. albicans which, like those of other fungi, only contains one set of chromosomes with one copy of each gene, but they can be mated to easily create the diploid form. “We used haploid C. albicans strains and replaced genes that we wanted to eliminate with a ‘gene drive’ that we previously developed and adjusted to the specific biology of C. albicans. After mating, these ‘selfish genetic elements’ proceed to replace the normal copy of the gene in the diploid fungi,” said Church, PhD, who is 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 new 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). After the targeted gene sequence has been cut out, 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 will also substitute the gene’s counterpart in the other chromosome, effectively deleting the original version from the organism entirely.

By applying their gene deletion approach, the team was able to identify combinations of genes that act synergistically in defying