Department of Life Sciences, Chalmers University of Technology, Gothenburg SE-41296, Sweden.
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
ACS Synth Biol. 2023 Aug 18;12(8):2278-2289. doi: 10.1021/acssynbio.2c00690. Epub 2023 Jul 24.
Directed evolution is a preferred strategy to improve the function of proteins such as enzymes that act as bottlenecks in metabolic pathways. Common directed evolution approaches rely on error-prone PCR-based libraries where the number of possible variants is usually limited by cellular transformation efficiencies. Targeted mutagenesis can advance directed evolution approaches and help to overcome limitations in library generation. In the current study, we aimed to develop a high-efficiency time-controllable targeted mutagenesis toolkit in the yeast by employing the CRISPR/Cas9 technology. To that end, we fused the dCas9 protein with hyperactive variants of adenine and cytidine deaminases aiming to create an inducible CRISPR-based mutagenesis tool targeting a specific DNA sequence with extended editing windows and high mutagenesis efficiency. We also investigated the effect of guide RNA multiplexing on the mutagenesis efficiency both phenotypically and on the DNA level.
定向进化是一种改进蛋白质功能的首选策略，例如作为代谢途径瓶颈的酶。常见的定向进化方法依赖易错 PCR 文库，其中可能的变体数量通常受细胞转化效率的限制。靶向诱变可以推进定向进化方法，并有助于克服文库生成的限制。在本研究中，我们旨在通过利用 CRISPR/Cas9 技术在酵母中开发一种高效的、时间可控的靶向诱变工具包。为此，我们将 dCas9 蛋白与腺嘌呤和胞嘧啶脱氨酶的超活性变体融合，旨在创建一种基于 CRISPR 的诱导性诱变工具，靶向特定 DNA 序列，具有扩展的编辑窗口和高诱变效率。我们还研究了向导 RNA 多重化对表型和 DNA 水平诱变效率的影响。
