Department of Bioengineering, Imperial College London, London, SW7 2AZ, UK.
Plant Sciences and the Bioeconomy, Rothamsted Research, West Common, Harpenden, Hertfordshire, AL5 2JQ, UK.
Commun Biol. 2023 Aug 18;6(1):858. doi: 10.1038/s42003-023-05202-5.
CRISPR/Cas9-based technologies are revolutionising the way we engineer microbial cells. One of the key advantages of CRISPR in strain design is that it enables chromosomal integration of marker-free DNA, eliminating laborious and often inefficient marker recovery procedures. Despite the benefits, assembling CRISPR/Cas9 editing systems is still not a straightforward process, which may prevent its use and applications. In this work, we have identified some of the main limitations of current Cas9 toolkits and designed improvements with the goal of making CRISPR technologies easier to access and implement. These include 1) A system to quickly switch between marker-free and marker-based integration constructs using both a Cre-expressing and standard Escherichia coli strains, 2) the ability to redirect multigene integration cassettes into alternative genomic loci via Golden Gate-based exchange of homology arms, 3) a rapid, simple in-vivo method to assembly guide RNA sequences via recombineering between Cas9-helper plasmids and single oligonucleotides. We combine these methodologies with well-established technologies into a comprehensive toolkit for efficient metabolic engineering using CRISPR/Cas9. As a proof of concept, we developed the YaliCraft toolkit for Yarrowia lipolytica, which is composed of a basic set of 147 plasmids and 7 modules with different purposes. We used the toolkit to generate and characterize a library of 137 promoters and to build a de novo strain synthetizing 373.8 mg/L homogentisic acid.
基于 CRISPR/Cas9 的技术正在彻底改变我们设计微生物细胞的方式。CRISPR 在菌株设计中的一个关键优势是，它能够实现无标记 DNA 的染色体整合，从而消除了繁琐且通常效率低下的标记回收程序。尽管有这些好处，但组装 CRISPR/Cas9 编辑系统仍然不是一个简单的过程，这可能会阻碍其使用和应用。在这项工作中，我们确定了当前 Cas9 工具包的一些主要限制，并进行了改进设计，旨在使 CRISPR 技术更容易获得和实施。这些改进包括：1）使用表达 Cre 的大肠杆菌菌株和标准大肠杆菌菌株，快速切换无标记和基于标记的整合构建体的系统；2）通过基于 Golden Gate 的同源臂交换，将多基因整合盒重新定向到替代基因组座的能力；3）一种通过 Cas9-helper 质粒和单链寡核苷酸之间的重组，快速、简单的体内方法来组装向导 RNA 序列。我们将这些方法与成熟的技术相结合，为使用 CRISPR/Cas9 进行高效代谢工程提供了一个全面的工具包。作为概念验证，我们为 Yarrowia lipolytica 开发了 YaliCraft 工具包，该工具包由一组基本的 147 个质粒和 7 个具有不同用途的模块组成。我们使用该工具包生成和表征了一个包含 137 个启动子的文库，并构建了一个从头开始的菌株，能够合成 373.8 mg/L 对羟基苯乙酸。
