Department of Molecular and Cellular Biology, University of Guelph, Guelph, Canada.
mSphere. 2024 Feb 28;9(2):e0076123. doi: 10.1128/msphere.00761-23. Epub 2024 Jan 24.
The overexpression of genes frequently arises in (formerly ) via gain-of-function mutations, gene duplication, or aneuploidies, with important consequences on pathogenesis traits and antifungal drug resistance. This highlights the need to develop specific genetic tools to mimic and study genetic amplification in this important fungal pathogen. Here, we report the development, validation, and applications of the first clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system in for targeted genetic overexpression. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in , and further assess optimal guide RNA targeting for robust overexpression. We demonstrate the applications of CRISPRa to overexpress genes involved in fungal pathogenesis and drug resistance and detect corresponding phenotypic alterations in these key traits, including the characterization of novel phenotypes. Finally, we capture strain variation using our CRISPRa system in two commonly used genetic backgrounds. Together, this tool will expand our capacity for functional genetic overexpression in this pathogen, with numerous possibilities for future applications.IMPORTANCE (formerly ) is an important fungal pathogen that is now the second leading cause of candidiasis infections. A common strategy that this pathogen employs to resist antifungal treatment is through the upregulation of gene expression, but we have limited tools available to study this phenomenon. Here, we develop, optimize, and apply the use of CRISPRa as a means to overexpress genes in . We demonstrate the utility of this system to overexpress key genes involved in antifungal susceptibility, stress tolerance, and biofilm growth. This tool will be an important contribution to our ability to study the biology of this important fungal pathogen.
基因的过表达通常是通过获得性功能突变、基因复制或非整倍体而产生的，这对发病机制特征和抗真菌药物耐药性有重要影响。这突出表明需要开发特定的遗传工具来模拟和研究这个重要真菌病原体中的基因扩增。在这里，我们报告了第一个在 中用于靶向基因过表达的成簇规律间隔短回文重复（CRISPR）激活（CRISPRa）系统的开发、验证和应用。使用该系统，我们证明了 CRISPRa 能够在 中驱动基因的高水平表达，并进一步评估了用于稳健过表达的最佳向导 RNA 靶向。我们展示了 CRISPRa 在过表达参与真菌发病机制和耐药性的基因中的应用，并在这些关键特征中检测到相应的表型改变，包括新型表型的特征。最后，我们使用我们的 CRISPRa 系统在两个常用的 遗传背景下捕获菌株变异。总之，该工具将扩大我们在该病原体中进行功能基因过表达的能力，为未来的应用提供了许多可能性。
（以前称为）是一种重要的真菌病原体，现在是导致念珠菌感染的第二大原因。该病原体用来抵抗抗真菌治疗的一种常见策略是上调基因表达，但我们可用的研究这种现象的工具有限。在这里，我们开发、优化并应用了 CRISPRa 在 中过表达基因的用途。我们证明了该系统在过表达参与抗真菌敏感性、应激耐受和生物膜生长的关键基因中的效用。该工具将是我们研究这个重要真菌病原体生物学能力的重要贡献。
