Fig. 1.

Design of a CRISPRa system for Candida albicans. a) The single-plasmid system contains a catalytically inactive (“dead”) dCas9 protein, paired with the VPR complex of activator domains to cause upregulation at a target locus. The sgRNA region can be subjected to Golden Gate cloning with a custom N20 sequence in order to target the dCas9-VPR complex to a desired region of DNA through complementary base-pairing. The PacI enzyme is used to linearize the plasmid at the NEUT5L locus, which allows the plasmid to be integrated into the cell's chromosome and remain in the cell following cell division. Panel created with BioRender.com. b) Once the sgRNA recognizes and binds to a target sequence of DNA, the dCas9-VPR complex will cause the recruitment of transcription initiation complex factors to the adjacent genetic material, thus increasing gene expression from that region. Panel created with BioRender.com. c) Wild-type C. albicans cells and cells containing a nontargeting CRISPRa plasmid were grown in YPD at 30°C to compare fitness under standard laboratory conditions. Growth was recorded via optical density at 600 nm at 15 min intervals over the course of 24 h. Data points show the mean and standard deviation at each time point (n = 32).