Background

C. albicans is a difficult organism to manipulate genetically. Since it normally exists as a diploid that does not readily undergo sexual reproduction, homozygous recessive mutations require sequential modification of each locus. The development and application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mutagenesis in C. albicans facilitates genetic manipulations because it allows simultaneous mutation of both alleles (Vyas et al., 2015; Min et al., 2016; Ng and Dean, 2017). CRISPR gene editing involves recruitment of an RNA-guided nuclease to a complementary target site adjacent to an NGG protospacer adjacent motif (PAM) (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013). CRISPR associated (Cas) nuclease is targeted with high specificity through complementary base pairing between a guide RNA associated with trans-activating CRISPR RNA (tracrRNA), which binds Cas9 (Gasiunas et al., 2012). Since the chromosomal target sequence is only ~20 nucleotides, expression of a single guide RNA (sgRNA) fused to a ~80 nucleotide tracrRNA, along with Cas9, is sufficient for targeted double-strand DNA cleavage. Since chromosomal breaks are lethal, there is a strong selective pressure for double stranded break repair. In C. albicans, co-expression of a donor repair fragment, containing homology to regions flanking the break, allows repair of the break by homologous recombination. Thus, appropriate design of the donor repair fragment allows introduction of sequence deletions, replacements or other chromosomal alterations.

Our previous studies demonstrated that a key factor contributing to high efficiency CRISPR mutagenesis of C. albicans genes relies on optimal gRNA expression (Ng and Dean, 2017). Toward this end, we created gRNA expression vectors that permit high levels of gRNA expression. The basis for this high-level expression is in part due to the presence of a strong, RNA polymerase II promoter (P_ADH1). This promoter drives the expression of an sgRNA flanked by a 5’ tRNA and a 3’ hepatitis delta virus (HDV) ribozyme RNA. The presence of these 5’ and 3’ flanking RNA sequences serve to promote efficient post-transcriptional processing to produce a mature sgRNA with precise ends. In the presence of an appropriate donor repair fragment, this increased sgRNA expression dramatically improves CRISPR/Cas mutagenesis in C. albicans. In practice, execution of mutagenesis is quite simple. The gRNA expression plasmid cloning relies on the annealing and ligation of two short gRNA encoding oligonucleotides into pre-cut vectors. Mutagenesis involves co-transformation of an sgRNA plasmid and the donor repair fragment into C. albicans strains that express codon-optimized Cas9 nuclease. Described below are detailed protocols for the design, synthesis, and cloning of gRNA oligonucleotides, healing fragment construction, yeast transformations, and mutant verification.