The most recent genome-editing system called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat system with associated protein 9-nuclease) was employed to delete four non-essential genes (i.e., Caeco1, Caidh1, Carom2, and Cataf10) individually to establish their gene functionality annotations in pathogen Candida albicans. The biological roles of these genes were investigated with respect to the cell wall integrity and biogenesis, calcium/calcineurin pathways, susceptibility of mutants towards temperature, drugs and salts. All the mutants showed increased vulnerability compared to the wild-type background strain towards the cell wall-perturbing agents, (antifungal) drugs and salts. All the mutants also exhibited repressed and defective hyphal growth and smaller colony size than control CA14. The cell cycle of all the mutants decreased enormously except for those with Carom2 deletion. The budding index and budding size also increased for all mutants with altered bud shape. The disposition of the mutants towards cell wall-perturbing enzymes disclosed lower survival and more rapid cell wall lysis events than in wild types. The pathogenicity and virulence of the mutants was checked by adhesion assay, and strains lacking rom2 and eco1 were found to possess the least adhesion capacity, which is synonymous to their decreased pathogenicity and virulence.

Colin Ratledge Center for Microbial Lipids School of Agriculture Engineering and Food Science Shandong University of Technology Zibo P R China Clinical Research Center Medical University of Bialystok Białystok Poland

Colin Ratledge Center for Microbial Lipids School of Agriculture Engineering and Food Science Shandong University of Technology Zibo P R China Department of Biology South Texas Center of Emerging Infectious Diseases University of Texas San Antonio USA

Department of Biosciences COMSATS University Islamabad Islamabad Campus Park Road Islamabad Pakistan

Key Laboratory of Meat Processing and Quality Control College of Food Sciences Nanjing Agriculture University Jiangsu P R China