Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1H 5N4, Canada.
Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA.
G3 (Bethesda). 2023 Feb 9;13(2). doi: 10.1093/g3journal/jkac301.
For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPR activation to overexpress genes involved in pathogenesis and drug susceptibility, and contribute toward the identification of novel phenotypes. Consequently, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.
对于真菌病原体白色念珠菌，通过多种基因组改变（如非整倍体和功能获得性突变）很容易发生遗传过表达，这对宿主适应性、毒力和抗真菌药物耐药性的进化有重要影响。鉴于过表达在白色念珠菌生物学中的重要作用，开发和利用能够分析真菌细胞中高表达基因的工具至关重要。在这里，我们描述了一种新型的基于单质粒的 CRISPR 激活（CRISPRa）平台的开发、优化和应用，该平台用于靶向白色念珠菌中的基因遗传过表达，它使用向导 RNA 将激活复合物靶向感兴趣基因的启动子区域，从而驱动该基因的转录表达。使用该系统，我们证明了 CRISPRa 能够在白色念珠菌中驱动高水平的基因表达，并且我们评估了最优的向导 RNA 靶向以实现稳健和组成型过表达。我们通过 RNA 测序进一步证明了该系统的特异性。我们强调了 CRISPR 激活在过表达参与发病机制和药物敏感性的基因方面的应用，并有助于鉴定新的表型。因此，该工具将促进白色念珠菌遗传过表达研究的广泛应用。
