Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York, USA.
mSphere. 2019 Feb 13;4(1):e00002-19. doi: 10.1128/mSphere.00002-19.
Fungal pathogens are emerging as an important cause of human disease, and is among the most common causative agents of fungal infections. Studying this fungal pathogen is of the utmost importance and necessitates the development of molecular technologies to perform comprehensive genetic and functional genomic analysis. Here, we designed and developed a novel clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for targeted genetic repression in We engineered a nuclease-dead Cas9 (dCas9) construct that, paired with a guide RNA targeted to the promoter of an endogenous gene, is capable of targeting that gene for transcriptional repression. We further optimized a favorable promoter locus to achieve repression and demonstrated that fusion of dCas9 to an Mxi1 repressor domain was able to further enhance transcriptional repression. Finally, we demonstrated the application of this CRISPRi system through genetic repression of the essential molecular chaperone This is the first demonstration of a functional CRISPRi repression system in , and this valuable technology will enable many future applications in this critical fungal pathogen. Fungal pathogens are an increasingly important cause of human disease and mortality, and is among the most common causes of fungal disease. Studying this important fungal pathogen requires a comprehensive genetic toolkit to establish how different genetic factors play roles in the biology and virulence of this pathogen. Here, we developed a CRISPR-based genetic regulation platform to achieve targeted repression of genes. This CRISPR interference (CRISPRi) technology exploits a nuclease-dead Cas9 protein (dCas9) fused to transcriptional repressors. The dCas9 fusion proteins pair with a guide RNA to target genetic promoter regions and to repress expression from these genes. We demonstrated the functionality of this system for repression in and show that we can apply this technology to repress essential genes. Taking the results together, this work presents a new technology for efficient genetic repression in , with important applications for genetic analysis in this fungal pathogen.
真菌病原体正在成为人类疾病的一个重要原因，而 是最常见的真菌病原体之一。研究这种真菌病原体至关重要，需要开发分子技术来进行全面的遗传和功能基因组分析。在这里，我们设计并开发了一种新型的成簇规律间隔短回文重复干扰（CRISPRi）系统，用于靶向遗传抑制 。我们构建了一种无核酸酶 Cas9（dCas9）构建体，与靶向内源性基因启动子的向导 RNA 配对，能够靶向该基因进行转录抑制。我们进一步优化了一个有利的启动子位点以实现抑制，并证明将 dCas9 融合到 Mxi1 抑制结构域能够进一步增强转录抑制。最后，我们通过对必需分子伴侣 进行遗传抑制来证明了该 CRISPRi 系统的应用。这是在 中首次证明功能性 CRISPRi 抑制系统，这项有价值的技术将为该关键真菌病原体的许多未来应用提供支持。真菌病原体是人类疾病和死亡的一个日益重要的原因，而 是最常见的真菌疾病原因之一。研究这种重要的真菌病原体需要一个全面的遗传工具包，以确定不同的遗传因素在该病原体的生物学和毒力中所起的作用。在这里，我们开发了一种基于 CRISPR 的遗传调控平台，以实现 基因的靶向抑制。这种 CRISPR 干扰（CRISPRi）技术利用融合到转录抑制剂上的无核酸酶 Cas9 蛋白（dCas9）。dCas9 融合蛋白与向导 RNA 配对，靶向遗传启动子区域，并抑制这些基因的表达。我们证明了该系统在 中的抑制功能，并表明我们可以将该技术应用于抑制必需基因。总之，这项工作为 在高效遗传抑制方面提供了一种新技术，对于该真菌病原体的遗传分析具有重要的应用价值。
