利用CRISPR-Cas9核糖核蛋白复合物在非模式植物病原菌musiva中建立基因组编辑工具。

IF 4.9 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Joanna Tannous, Cole Sawyer, Md Mahmudul Hassan, Jesse L Labbe, Carrie Eckert
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引用次数: 0

摘要

CRISPR-Cas9是一种多功能基因组编辑系统,自2013年以来被广泛用于向模型和非模型物种的基因组中引入位点特异性修饰。这项技术被用于各种应用,从基因敲除、敲入、过表达到更精确的改变,例如在目标位点引入核苷酸。与锌指核酸酶和转录激活因子样效应核酸酶等先前的基因编辑策略相比,CRISPR-Cas9已被证明易于在新种中建立,并且具有高效率和特异性。丝状真菌中新兴的CRISPR-Cas9工具面临的巨大挑战是为非模式生物开发有效的转化方法。本文利用在模式物种里氏木霉(Trichoderma reesei)中建立的基于Cas9/sgRNA核糖核蛋白复合物(RNPs)的CRISPR-Cas9基因组编辑工具,开发了首个高效转化非模式物种Sphaerulina musiva的方案。这种真菌病原体对用于生物燃料生产的基础生物能源作物杨树属构成了真正的威胁。在这里,我们强调了设计sgrna及其计算验证的一般考虑因素。我们还描述了在这两个物种中使用分离的原生质体递送CRISPR-Cas9 RNP组件以及筛选靶向基因组编辑事件。musiva工程工具的开发可用于研究涉及多种过程的基因,如次级代谢、建立和致病性等,但也可用于开发遗传缓解方法。本文描述的方法为其他非模式孢子子囊菌转化系统的潜在发展提供了指导。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Establishment of a genome editing tool using CRISPR-Cas9 ribonucleoprotein complexes in the non-model plant pathogen <i>Sphaerulina musiva</i>.

Establishment of a genome editing tool using CRISPR-Cas9 ribonucleoprotein complexes in the non-model plant pathogen <i>Sphaerulina musiva</i>.

Establishment of a genome editing tool using CRISPR-Cas9 ribonucleoprotein complexes in the non-model plant pathogen <i>Sphaerulina musiva</i>.

Establishment of a genome editing tool using CRISPR-Cas9 ribonucleoprotein complexes in the non-model plant pathogen Sphaerulina musiva.

CRISPR-Cas9 is a versatile genome editing system widely used since 2013 to introduce site-specific modifications into the genomes of model and non-model species. This technology is used in various applications, from gene knock-outs, knock-ins, and over-expressions to more precise changes, such as the introduction of nucleotides at a targeted locus. CRISPR-Cas9 has been demonstrated to be easy to establish in new species and highly efficient and specific compared to previous gene editing strategies such as Zinc finger nucleases and transcription activator-like effector nucleases. Grand challenges for emerging CRISPR-Cas9 tools in filamentous fungi are developing efficient transformation methods for non-model organisms. In this paper, we have leveraged the establishment of CRISPR-Cas9 genome editing tool that relies on Cas9/sgRNA ribonucleoprotein complexes (RNPs) in the model species Trichoderma reesei and developed the first protocol to efficiently transform the non-model species, Sphaerulina musiva. This fungal pathogen constitutes a real threat to the genus Populus, a foundational bioenergy crop used for biofuel production. Herein, we highlight the general considerations to design sgRNAs and their computational validation. We also describe the use of isolated protoplasts to deliver the CRISPR-Cas9 RNP components in both species and the screening for targeted genome editing events. The development of engineering tools in S. musiva can be used for studying genes involved in diverse processes such as secondary metabolism, establishment, and pathogenicity, among many others, but also for developing genetic mitigation approaches. The approach described here provides guidance for potential development of transformation systems in other non-model spore-bearing ascomycetes.

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