Although the CRISPR/Cas9 gene-editing system can manipulate the genomes of many animals, plants, and fungi, it has difficulty coming to grips with Candida albicans, an exceptionally shifty foe. C. albicans has more moves than most gene-editing systems can keep up with. It has no plasmid system. It lacks any known meiotic phase. And it is diploid. Actually, it has a disturbing tendency toward aneuploidy. Thus, C. albicans is not only unpredictable and variable in matters chromosomal, it also maintains a protective redundancy.
C. albicans shrugs off conventional means of gene editing because it usually has two copies of every gene. Both copies of any particular gene are difficult to knock down efficiently enough to produce any phenotypic effect. And so it is hard to identify essential genes that could direct drug developers to the organism’s weaknesses. Finding C. albicans’ vulnerabilities is essential if this yeast, which is usually harmlessly commensal, is to be kept from causing deadly mucosal and systemic infections in immunocompromised people.
The problem of genomically seizing C. albicans was attacked by scientists at the Whitehead Institute. These scientists, led by Gerald R. Fink, Ph.D., a Whitehead founding member and a professor at MIT, optimized the CRISPR/Cas9 gene-editing system for C. albicans by recoding the Cas9 gene. The scientists described their innovation April 3 in the journal Science Advances, in an article entitled, “A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families.”
“The Cas9 gene had to be recoded because the leucine CUG codon is predominantly translated asserine, there are no autonomously replicating plasmids, and there are no expression systems for small RNAs,” the article’s authors wrote. “To express a Candida-compatible Cas9, we synthesized a Candida/Saccharomyces codon–optimized version of Cas9 (CaCas9) that avoids the use of the CUG codon, ensuring compatibility with all CTG clade species.”
The writers explained how they created two systems—a “Duet” system that requires the sequential integration of two plasmids, and a “Solo” system that consolidates the CRISPR system with the sgRNA system by fusing them in a single plasmid construct. “Both the Duet and Solo systems feature simplified ligation of annealed oligos into the site created with BsmBI, leaving no extraneous sequences,” the authors asserted.
Using their modified CRISPR/Cas9 gene-editing system in both laboratory and clinical strains, the Whitehead Institute scientists efficiently mutated in a single experiment both copies of several different genes, including members of a gene family important for antibiotic resistance as well as an essential gene. The scientists estimated that their CRISPR/Cas system should be able to target more than 98% of C. albicans' genome. That means it should be possible to determine which of C. albicans' 6,000 genes are essential and might make good drug targets.
“The improvement efficiency brought by this system expands the scale at which we can do genetics in this important pathogen,” said Valmik Vyas, Ph.D., a postdoctoral researcher in Dr. Fink’s laboratory. “It's an exciting time to be working on Candida.”
The scientists anticipate their system can overcome a key impediment to making multiple knockouts while analyzing gene families—the need for simultaneous mutation at multiple loci. The Candida genome, the scientists pointed out, is populated by many gene families, including more than 120 drug efflux pumps. This redundancy impedes analysis of the resistance to antifungal agents because the construction of multiple mutations in the members of these families is beyond current technology. These pumps also give Candida a high inherent drug resistance, rendering all but one drug resistance marker useless.
“[Our] system permits the creation of strains with mutations in multiple genes, gene families, and genes that encode essential functions,” concluded the authors. “This CRISPR system is also effective in a fresh clinical isolate of undetermined ploidy. Our method transforms the ability to manipulate the genome of Candida and provides a new window into the biology of this pathogen.”