ACtivE: Assembly and CRISPR-Targeted in Vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time.
ACtivE: Assembly and CRISPR-Targeted in Vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time.
复制标题
ACTIVE：使用最少的试剂和时间进行酵母基因组工程的组装和CRISPR靶向体内编辑。
DOI：
10.1021/acssynbio.2c00175
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发表时间：
2022-11-18
影响因子：
4.7
通讯作者：
Rios-Solis, Leonardo
中科院分区：
文献类型：
--
作者：
Malci, Koray;Jonguitud-Borrego, Nestor;Waillet, Hugo van der Straten;Puodziunaite, Urte;Johnston, Emily J.;Rosser, Susan J.;Rios-Solis, Leonardo
关键词：
由于其复杂性，CRISPR/Cas系统已经成为一种广泛使用的酵母基因组编辑方法。然而，CRISPR方法通常依赖于预先组装的DNA和额外的克隆步骤来传递gRNA、Cas蛋白和供体DNA。这些费力的步骤可能会阻碍其有效性。在这里，我们提出了另一种方法，组装和CRISPR-目标体内编辑(主动)，只依赖于在体组装线性DNA片段的质粒和供体DNA构建。因此，根据用户的需要，这些部分可以很容易地从存储库中选择和组合，作为快速基因组编辑的工具包，而不需要任何昂贵的试剂。该工具包包含经过验证的线性DNA片段，便于在室温下存储、共享和运输，大大降低了昂贵的运输成本和组装时间。在优化这一技术后，酵母基因组中自主复制序列(ARS)附近的8个基因座也在整合和基因表达效率以及这些区域的中断对细胞适合性的影响方面进行了表征。通过构建β-胡萝卜素途径，展示了活性物质的灵活性和多重作用能力。在短短几天的时间里，在酿酒酵母BY4741上从头开始获得了80%的单基因整合效率和50%的三重整合效率，而不需要使用体外DNA组装方法、限制性内切酶或额外的克隆步骤。这项研究提供了一种易于使用的标准化方法来加速酵母基因组工程，并为酵母合成生物学和代谢工程目的提供了明确的基因组位置选择。
Thanks to its sophistication, the CRISPR/Cas system has been a widely used yeast genome editing method. However, CRISPR methods generally rely on preassembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose an alternative method, Assembly and CRISPR-targeted in vivo Editing (ACtivE), that only relies on in vivo assembly of linear DNA fragments for plasmid and donor DNA construction. Thus, depending on the user’s need, these parts can be easily selected and combined from a repository, serving as a toolkit for rapid genome editing without any expensive reagent. The toolkit contains verified linear DNA fragments, which are easy to store, share, and transport at room temperature, drastically reducing expensive shipping costs and assembly time. After optimizing this technique, eight loci proximal to autonomously replicating sequences (ARS) in the yeast genome were also characterized in terms of integration and gene expression efficiencies and the impacts of the disruptions of these regions on cell fitness. The flexibility and multiplexing capacity of the ACtivE were shown by constructing a β-carotene pathway. In only a few days, >80% integration efficiency for single gene integration and >50% integration efficiency for triplex integration were achieved on Saccharomyces cerevisiae BY4741 from scratch without using in vitro DNA assembly methods, restriction enzymes, or extra cloning steps. This study presents a standardizable method to be readily employed to accelerate yeast genome engineering and provides well-defined genomic location alternatives for yeast synthetic biology and metabolic engineering purposes.
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影响因子：
3.7
作者：
Engler, Carola;Kandzia, Romy;Marillonnet, Sylvestre
通讯作者：
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