Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System.
Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System.
复制标题
DOI：
10.1128/msphere.00130-16
复制
发表时间：
2016-05
期刊：
影响因子：
4.8
通讯作者：
Mitchell AP
中科院分区：
文献类型：
--
作者：
Min K;Ichikawa Y;Woolford CA;Mitchell AP
真菌白色念珠菌是主要病原体。该生物体的遗传分析揭示了致病性、耐药性和其他独特生物学特征的决定因素，以及潜在药物靶标的身份。最近，通过 Vyas 等人实施的 CRISPR 基因组编辑技术，靶向突变的创建大大加速了。 [科学进展 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]。在这项研究中，我们发现 CRISPR 元件可以从仅短暂存在的基因中表达，并且我们开发了一种瞬时 CRISPR 系统，可以进一步加速白色念珠菌的基因操作。成簇规则间隔短回文重复序列 (CRISPR) 和 CRISPR 相关基因 9 (CRISPR-Cas9) 系统广泛用于从真菌到植物和动物等生物体的基因组编辑应用。最近，针对二倍体真菌病原体白色念珠菌开发了CRISPR-Cas9系统；该系统极大地加速了基因操作[V. K. Vyas、M. I. Barrasa 和 G. R. Fink，Sci Adv 1(3):e1500248，2015，http://dx.doi.org/10.1126/sciadv.1500248]。我们在此表明，CRISPR-Cas9 遗传元件可以短暂发挥作用，无需稳定整合到基因组中，从而能够引入基因删除构建体。我们描述了一种瞬时 CRISPR-Cas9 系统，可在白色念珠菌中高效进行基因删除。我们的观察表明，有两种机制导致纯合性缺失：（i）将DNA转化为每个等位基因的独立重组和（ii）将DNA转化为一个等位基因的重组，然后对第二个等位基因进行基因转化。我们的方法将简化白色念珠菌的基因功能分析，我们的结果表明 DNA 在该生物体转化后可以短暂发挥作用。重要性 白色念珠菌是一种主要病原体。该生物体的遗传分析揭示了致病性、耐药性和其他独特生物学特征的决定因素，以及潜在药物靶标的身份。最近，通过 Vyas 等人实施的 CRISPR 基因组编辑技术，靶向突变的创建大大加速了。 [科学进展 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]。在这项研究中，我们发现 CRISPR 元件可以从仅短暂存在的基因中表达，并且我们开发了一种瞬时 CRISPR 系统，可以进一步加速白色念珠菌的基因操作。
The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation. Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C. albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C. albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation.