Development of CRISPR-Cas9 ribonucleoprotein-based method for ECE1 disruption and functional study in Candida albicans with Streptococcus salivarius K12 [EMBARGOED]

Abstract

ECE1 encodes the toxic fungal protein candidalysin, which is integral to mucosal infections by Candida albicans. The role of ECE1 in C. albicans virulence in the planktonic state is known, but its role in the more virulent biofilm state has not been studied. Methods A ribonucleoprotein-based CRISPR-Cas9 method for generation of C. albicans ECE1 knockout strain, ece1?/? was developed, involving design of donor DNA (dDNA), selection of guide RNA (gRNA) targets, Cas9 to gRNA ratio and transformation methods. C. albicans SC5314 was used in all transformation experiments. ece1?/? was compared against SC5314 for C. albicans aggregation, biofilm formation, dimorphism, and immunomodulation in host cells. ECE1 was studied in C. albicans monoculture biofilm, and dual-culture with the probiotic S. salivarius K12. Results The dDNA design was a linear double-stranded DNA fragment containing a C. albicans codon-optimized CaNAT1 nourseothricin selection marker and 500bp flanking homology arms for insertion into the ECE1 locus; use of plasmid vectors for delivery of the selection marker resulted in unstable expression. Delivery of the dDNA into C. albicans was most effective using electroporation. Among the three gRNAs (sgRNA3’, sgRNAmid and sgRNA5’) selected, in vitro cleavage assay that showed sgRNA5’ and sgRNAmid had highest targeting efficiency. For CRISPR-Cas9 RNP delivery experiments, using sgRNAmid at Cas9 to sgRNA ratio of 1:3 resulted in highest transformation efficiency (49 colonies/µg). The HDR dDNA was integrated into the C. albicans genome to stably express nourseothricin resistance. However, colony PCR and sequencing results indicate ECE1 was not knocked out and replaced with knock-in of the dDNA, suggesting off-target integration of the dDNA. C. albicans aggregation, biofilm formation and dimorphism were significantly (p<0.05) different in ece1?/? compared to SC5314. However, biofilm supernatant from ECE1 did not significantly affect proliferation, migration and IL-6 and IL-8 immunomodulation in KD oral cells (p>0.05). S. salivarius K12 did not affect ECE1 function in C. albicans across all virulence traits tested.