Using CRISPR-Cas9 to Study the Evolutionary Role of Genome Instability in Candida albicans

Candida albicans is a typical member of the human microbiome which can also cause a range of infections from superficial to systemic. The ability of C. albicans to survive the broad range of conditions it encounters in the human host is partially attributed to its genomic instability. This instability generates diversity and allows selection of fitter genotypes which can thrive in the hostile host microenvironments. Consequently, C. albicans clinical isolates have diverse karyotypes. C. albicans chromosomal rearrangements often occur around repetitive elements including the Major Repeat Sequence (MRS), a conserved repeat array of unknown function occurring on seven of the eight C. albicans chromosomes. We hypothesise that MRS elements serve as instability hotspots to facilitate genomic rearrangements and rapid evolution.

This study aims to establish a cause-and-effect relationship between MRS-driven chromosome rearrangements and generation of novel, fitter genotypes. To this end, we have used a CRISPR-Cas9 approach to generate double strand breaks within the MRS, inducing chromosome rearrangements in unstressed standard laboratory growth conditions. These unstable strains have then been evolved in clinically relevant stresses, including antifungal drugs.

Long read sequencing shows numerous translocations have been successfully generated around the MRS in these unstable strains. CRISPR-Cas9 can therefore be used to generate chromosomal rearrangements in C. albicans. Strains bearing translocations had morphological and fitness changes, indicating that rearrangements at the MRS can generate diversity. Evolution experiments showed that MRS-destabilized strains had higher genome instability than WT C. albicans, undergoing significantly more rearrangements throughout. In one instance, this increased plasticity resulted in faster adaptation to stress.

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This study demonstrates the ability to generate karyotypic alterations in C. albicans using CRISPR-Cas9. Furthermore, it supports the evolutionary role of the MRS as a facilitator of genome plasticity, helping to generate karyotypic diversity. There is some evidence that this enables Candida to adapt to environmental stress, but this requires further investigation.