Use of RNA-Protein Complexes for Genome Editing in Non-albicans Candida Species.
Use of RNA-Protein Complexes for Genome Editing in Non-albicans Candida Species.
复制标题
DOI：
10.1128/msphere.00218-17
复制
发表时间：
2017-05
期刊：
影响因子：
4.8
通讯作者：
Hogan DA
中科院分区：
文献类型：
--
作者：
Grahl N;Demers EG;Crocker AW;Hogan DA
用于白色念珠菌的现有CRISPR-Cas9基因组修饰系统依赖于构建体内源性表达Cas9蛋白和指导RNA，由于启动子活性不足，在其他念珠菌属物种中不能有效地工作。在这里，我们提出了一种表达免费的方法，使用RNA-蛋白质复合物，并证明其在三个念珠菌属的耐药谱已知的使用。我们建议，该系统将有助于真菌，缺乏既定的遗传系统的遗传分析。规则间隔短回文重复序列（CRISPR）-Cas9基因组修饰系统极大地促进了真菌病原体的遗传分析。 在设计用于白色念珠菌的CRISPR-Cas9基因组编辑方法中，编码必要组分的DNA在靶细胞中表达。不幸的是，在白色念珠菌中有效工作的表达构建体不一定在念珠菌属或相关的棒孢菌属内的其他致病性物种中良好表达。为了避免对物种特异性表达构建体的需求，我们实施了一种无表达的CRISPR基因组编辑系统，并证明了其在三种不同的非白色念珠菌物种中的成功使用：Candida（Clavispora）lusitaniae，Candida glabrata和Candida auris。在CRISPR-Cas9介导的基因组编辑方法中，靶向的双链DNA断裂可以通过同源重组到研究者设计的模板来修复。在该方案中，在转化与基因特异性RNA和支架RNA复合的纯化的Cas9蛋白（称为RNA-蛋白质复合物（RNP））时诱导DNA切割。在所有三个物种中，RNP的使用增加了转化体的数量和其中靶基因被选择标记成功替换的转化体的百分比。我们构建了已知的或假定的过氧化氢酶基因缺陷的突变体在C. lusitaniae，C. glabrata，和C. auris，并证明，在所有三个物种中，突变体比亲本菌株更容易受到过氧化氢。这种方法避免了表达CRISPR-Cas9组分的需要，可广泛用于研究遗传工具有限的各种念珠菌属物种和新出现的病原体。用于白色念珠菌的现有CRISPR-Cas9基因组修饰系统依赖于构建体内源性表达Cas9蛋白和指导RNA，由于启动子活性不足，在其他念珠菌属物种中不能有效工作。在这里，我们提出了一种表达免费的方法，使用RNA-蛋白质复合物，并证明其在三个念珠菌属的耐药谱已知的使用。我们建议，该系统将有助于真菌，缺乏既定的遗传系统的遗传分析。
Existing CRISPR-Cas9 genome modification systems for use in Candida albicans, which rely on constructs to endogenously express the Cas9 protein and guide RNA, do not work efficiently in other Candida species due to inefficient promoter activity. Here, we present an expression-free method that uses RNA-protein complexes and demonstrate its use in three Candida species known for their drug resistance profiles. We propose that this system will aid the genetic analysis of fungi that lack established genetic systems. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome modification systems have greatly facilitated the genetic analysis of fungal pathogens. In CRISPR-Cas9 genome editing methods designed for use in Candida albicans, DNAs that encode the necessary components are expressed in the target cells. Unfortunately, expression constructs that work efficiently in C. albicans are not necessarily expressed well in other pathogenic species within the genus Candida or the related genus Clavispora. To circumvent the need for species-specific expression constructs, we implemented an expression-free CRISPR genome editing system and demonstrated its successful use in three different non-albicans Candida species: Candida (Clavispora) lusitaniae, Candida glabrata, and Candida auris. In CRISPR-Cas9-mediated genome editing methods, a targeted double-stranded DNA break can be repaired by homologous recombination to a template designed by the investigator. In this protocol, the DNA cleavage is induced upon transformation of purified Cas9 protein in complex with gene-specific and scaffold RNAs, referred to as RNA-protein complexes (RNPs). In all three species, the use of RNPs increased both the number of transformants and the percentage of transformants in which the target gene was successfully replaced with a selectable marker. We constructed mutants defective in known or putative catalase genes in C. lusitaniae, C. glabrata, and C. auris and demonstrated that, in all three species, mutants were more susceptible to hydrogen peroxide than the parental strain. This method, which circumvents the need for expression of CRISPR-Cas9 components, may be broadly useful in the study of diverse Candida species and emergent pathogens for which there are limited genetic tools. IMPORTANCE Existing CRISPR-Cas9 genome modification systems for use in Candida albicans, which rely on constructs to endogenously express the Cas9 protein and guide RNA, do not work efficiently in other Candida species due to inefficient promoter activity. Here, we present an expression-free method that uses RNA-protein complexes and demonstrate its use in three Candida species known for their drug resistance profiles. We propose that this system will aid the genetic analysis of fungi that lack established genetic systems.