Norton Emily L, Sherwood Racquel K, Bennett Richard J
Department of Molecular Microbiology and Immunology, Brown University, Providence, Rhode Island, USA.
mSphere. 2017 Jun 21;2(3). doi: 10.1128/mSphere.00217-17. eCollection 2017 May-Jun.
is a member of the clade that includes a diverse group of fungal species relevant to both human health and biotechnology. This species exhibits a full sexual cycle to undergo interconversion between haploid and diploid forms. is also an emerging opportunistic pathogen that can cause serious bloodstream infections in the clinic and yet has often proven to be refractory to facile genetic manipulations. In this work, we develop a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (Cas9) system to enable genome editing of . We demonstrate that expression of CRISPR-Cas9 components under species-specific promoters is necessary for efficient gene targeting and can be successfully applied to multiple genes in both haploid and diploid isolates. Gene deletion efficiencies with CRISPR-Cas9 were further enhanced in strains lacking the established nonhomologous end joining (NHEJ) factors Ku70 and DNA ligase 4. These results indicate that NHEJ plays an important role in directing the repair of DNA double-strand breaks (DSBs) in and that removal of this pathway increases integration of gene deletion templates by homologous recombination. The described approaches significantly enhance the ability to perform genetic studies in, and promote understanding of, this emerging human pathogen and model sexual species. The ability to perform efficient genome editing is a key development for detailed mechanistic studies of a species. is an important member of the clade and is relevant both as an emerging human pathogen and as a model for understanding mechanisms of sexual reproduction. We highlight the development of a CRISPR-Cas9 system for efficient genome manipulation in and demonstrate the importance of species-specific promoters for expression of CRISPR components. We also demonstrate that the NHEJ pathway contributes to non-template-mediated repair of DNA DSBs and that removal of this pathway enhances efficiencies of gene targeting by CRISPR-Cas9. These results therefore establish important genetic tools for further exploration of biology.
是包括与人类健康和生物技术相关的多种真菌物种的进化枝的成员。该物种表现出完整的有性周期，可在单倍体和二倍体形式之间进行相互转换。它也是一种新兴的机会性病原体，可在临床上引起严重的血流感染，但常常被证明难以进行简便的基因操作。在这项工作中，我们开发了一种成簇规律间隔短回文重复序列（CRISPR）和CRISPR相关基因9（Cas9）系统，以实现对其基因组的编辑。我们证明，在物种特异性启动子下表达CRISPR-Cas9组件对于高效基因靶向是必要的，并且可以成功应用于单倍体和二倍体分离株中的多个基因。在缺乏已确定的非同源末端连接（NHEJ）因子Ku70和DNA连接酶4的菌株中，CRISPR-Cas9的基因缺失效率进一步提高。这些结果表明，NHEJ在指导DNA双链断裂（DSB）的修复中起重要作用，并且去除该途径可通过同源重组增加基因缺失模板的整合。所描述的方法显著增强了对这种新兴人类病原体和有性生殖模型物种进行遗传研究的能力，并促进了对其的理解。进行高效基因组编辑的能力是对一个物种进行详细机制研究的关键进展。是进化枝的重要成员，作为新兴的人类病原体以及理解有性生殖机制的模型都具有重要意义。我们强调了开发用于在中进行高效基因组操作的CRISPR-Cas9系统，并证明了物种特异性启动子对CRISPR组件表达的重要性。我们还证明，NHEJ途径有助于DNA DSB的非模板介导修复，并且去除该途径可提高CRISPR-Cas9的基因靶向效率。因此，这些结果为进一步探索生物学建立了重要的遗传工具。
