Rapid Hypothesis Testing in Candida albicans Clinical Isolates Using a Cloning-Free, Modular, and Recyclable System for CRISPR-Cas9 Mediated Mutant and Revertant Construction

As increasing evidence emerges that interstrain genetic diversity among Candida albicans clinical isolates underpins phenotypic variation compared to the reference isolate SC5314, new genetic tools are required to interrogate gene function across strain backgrounds. Here, the -flipper plasmid was reengineered to contain a C. albicans codon optimized hygromycin B resistance gene ( ). Cassettes were PCR-amplified from both -flipper and -flipper plasmids using primers with homologous sequences flanking target genes of interest to serve as repair templates. Ribonucleoprotein (RNP) complexes containing proprietary CRISPR RNAs (crRNAs), universal transactivating CRISPR RNA (tracrRNA), and Cas9 protein were assembled and transformed, along with both repair templates, by electroporation into C. albicans. Homozygous deletion of the gene results in red-pigmented colonies and this gene was used to validate our approach. Both in SC5314 and a variety of clinical isolates (529L, JS15, SJCA1, TW1), homozygous gene targeting was nearly 100% when plating on media containing nourseothricin and hygromycin B with transformation efficiencies exceeding 10 homozygous deletion mutants per μg of DNA. A gene reversion system was also employed with plasmids pDUP3 and pDIS3 engineered to contain the terminator and an overlap extension PCR-mediated approach combined with CRISPR-Cas9 targeting at the neutral locus. A variety of single or compound mutants (Δ/Δ , Δ/Δ Δ/Δ , Δ/Δ ) and their revertant strains were constructed and phenotypically validated by a variety of assays, including biofilm formation, hyphal growth, and macrophage IL-1β response. Thus, we have established a cloning-free, modular system for highly efficient homozygous gene deletion and reversion in diverse isolates. Recently, phenotypic heterogeneity in Candida albicans isolates has been recognized as an underappreciated factor contributing to gene diversification and broadly impacts strain-to-strain antifungal resistance, fitness, and pathogenicity. We have designed a cloning-free genetic system for rapid gene deletion and reversion in C. albicans clinical isolates that interlaces established recyclable genetic systems with CRISPR-Cas9 technology. The -flipper was reengineered to contain encoding resistance to hygromycin B. Using a modular PCR-mediated approach coupled with ribonucleoprotein assembly with commercial reagents, both - and -flipper cassettes were simultaneously integrated at loci with high efficiency (10