Institute for Bioengineering, School of Engineering, University of Edinburgh, EdinburghEH9 3BF, U.K.
Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, EdinburghEH9 3BD, U.K.
ACS Synth Biol. 2022 Nov 18;11(11):3629-3643. doi: 10.1021/acssynbio.2c00175. Epub 2022 Oct 17.
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 Editing (ACtivE), that only relies on 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 BY4741 from scratch without using 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.
得益于其复杂性，CRISPR/Cas 系统已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递呈 gRNA、Cas 蛋白和供体 DNA。这些繁琐的步骤可能会阻碍其应用。在这里，我们提出了一种替代方法，即组装和 CRISPR 靶向编辑（ACtivE），该方法仅依赖于线性 DNA 片段的组装来构建质粒和供体 DNA。因此，根据用户的需求，这些部分可以从存储库中轻松选择和组合，作为一种无需昂贵试剂的快速基因组编辑工具包。该工具包包含经过验证的线性 DNA 片段，易于在室温下储存、共享和运输，大大降低了昂贵的运输成本和组装时间。在优化该技术后，还对酵母基因组中靠近自主复制序列 (ARS) 的八个基因座的整合和基因表达效率以及这些区域的破坏对细胞适应性的影响进行了特征描述。通过构建β-胡萝卜素途径，展示了 ACtivE 的灵活性和多路复用能力。在短短几天内，无需使用 DNA 组装方法、限制酶或额外的克隆步骤，就可以从零开始在 BY4741 中实现单基因整合的>80%整合效率和三联体整合的>50%整合效率。本研究提供了一种可标准化的方法，可用于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替代方案。
