CRISPR/Cas9 Ribonucleoprotein-Mediated Mutagenesis in Sporisorium reilianum.

IF 16.4 1区化学 Q1 CHEMISTRY, MULTIDISCIPLINARY

Accounts of Chemical Research Pub Date : 2024-04-20 DOI:10.21769/BioProtoc.4978

Janina Werner, Weiliang Zuo, Gunther Doehlemann

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引用次数: 0

Abstract

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has become the state of the art for mutagenesis in filamentous fungi. Here, we describe a ribonucleoprotein complex (RNP)-mediated CRISPR/Cas9 for mutagenesis in Sporisorium reilianum. The efficiency of the method was tested in vitro with a cleavage assay as well as in vivo with a GFP-expressing S. reilianum strain. We applied this method to generate frameshift- and knock-out mutants in S. reilianum without a resistance marker by using an auto-replicating plasmid for selection. The RNP-mediated CRISPR/Cas9 increased the mutagenesis efficiency, can be applied for all kinds of mutations, and enables a marker-free genome editing in S. reilianum. Key features • First CRISPR/Cas9 application in S. reilianum. • Generation of S. reilianum mutants without genomic integration of resistance marker. • Allows the generation of multiple gene knockouts as well as deletion of large genomic regions.

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Reilianum 孢子虫中 CRISPR/Cas9 核糖核蛋白介导的突变。

聚类规则间隔短回文重复序列/CRISPR相关蛋白9（CRISPR/Cas9）已成为丝状真菌诱变的最新技术。在这里，我们描述了一种由核糖核蛋白复合物（RNP）介导的CRISPR/Cas9诱变Sporisorium reilianum的方法。我们在体外用裂解试验测试了这种方法的效率，并在体内用表达 GFP 的雷氏孢子菌株进行了测试。我们利用这种方法，通过使用自动复制质粒进行选择，在 S. reilianum 中产生了无抗性标记的移帧突变体和基因敲除突变体。RNP 介导的 CRISPR/Cas9 提高了诱变效率，可用于所有类型的突变，并实现了 S. reilianum 的无标记基因组编辑。主要特点 - 首次在 S. reilianum 中应用 CRISPR/Cas9。- 在不整合抗性标记的情况下生成 S. reilianum 突变体。- 允许产生多基因敲除以及大基因组区域的删除。

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来源期刊

Accounts of Chemical Research 化学-化学综合

CiteScore

31.40

自引率

1.10%

发文量

312

审稿时长

2 months

期刊介绍： Accounts of Chemical Research presents short, concise and critical articles offering easy-to-read overviews of basic research and applications in all areas of chemistry and biochemistry. These short reviews focus on research from the author’s own laboratory and are designed to teach the reader about a research project. In addition, Accounts of Chemical Research publishes commentaries that give an informed opinion on a current research problem. Special Issues online are devoted to a single topic of unusual activity and significance. Accounts of Chemical Research replaces the traditional article abstract with an article "Conspectus." These entries synopsize the research affording the reader a closer look at the content and significance of an article. Through this provision of a more detailed description of the article contents, the Conspectus enhances the article's discoverability by search engines and the exposure for the research.