CRISPR-Cas9 Cytidine-Base-Editor Mediated Continuous In Vivo Evolution in Aspergillus nidulans.

IF 3.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS ACS Synthetic Biology Pub Date : 2025-01-26 DOI:10.1021/acssynbio.4c00716

Yuan Tian, Qing Xu, Meng Pang, Youchu Ma, Zhiruo Zhang, Dongfang Zhang, Donghui Guo, Lupeng Wang, Qingbin Li, Yanling Li, Fanglong Zhao

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Abstract

Filamentous fungi are important cell factories for producing chemicals, organic acids, and enzymes. Although several genome editing tools are available for filamentous fungi, few effectively enable continuous evolution for rational engineering of complex phenotype. Here, we present CRISPR-Cas9 cytidine-base-editor (CBE) assisted in vivo evolution by continuously delivering a combinatorial sgRNA library to filamentous fungi. The method was validated by targeting core genes of 46 natural product biosynthetic gene clusters in Aspergillus nidulans NRRL 8112 to eliminate fungal toxins via six rounds of evolution. NGS analysis revealed the average C-to-T conversion rates in the first, third, and sixth rounds were 2.02%, 5.25%, and 9.34%, respectively. Metabolic profiles of the evolved mutants exhibited significant changes, allowing for the isolation of clean-background strains with enhanced production of an antifungal compound Echinocandin B. This study demonstrates that CBE-mediated in vivo evolution greatly facilitates the iterative refinement of complex morphogenetic traits in filamentous fungi.

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丝状真菌是生产化学品、有机酸和酶的重要细胞工厂。虽然有几种基因组编辑工具可用于丝状真菌，但很少有工具能有效地实现持续进化，以合理地设计复杂的表型。在这里，我们介绍了 CRISPR-Cas9 细胞苷碱基编辑器（CBE）通过向丝状真菌持续传递组合 sgRNA 文库来辅助体内进化。该方法通过靶向裸曲霉（Aspergillus nidulans NRRL 8112）46个天然产物生物合成基因簇的核心基因，通过六轮进化消除真菌毒素进行了验证。NGS 分析显示，第一轮、第三轮和第六轮的平均 C-T 转换率分别为 2.02%、5.25% 和 9.34%。进化突变体的代谢特征发生了显著变化，从而分离出了背景清洁的菌株，其抗真菌化合物棘白菌素 B 的产量得到了提高。

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.