Yuzbashev Tigran, Yuzbasheva Evgeniya, Melkina Olga, Patel Davina, Bubnov Dmitrii, Dietz Heiko, Ledesma-Amaro Rodrigo
Rothamsted Research.
BioMediCan Inc.
Res Sq. 2023 Apr 4:rs.3.rs-2738543. doi: 10.21203/rs.3.rs-2738543/v1.
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 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 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 generated and characterized a library of 137 promoters and built a strain synthetizing 373.8 mg/L homogentisic acid.
基于CRISPR/Cas9的技术正在彻底改变我们对微生物细胞进行工程改造的方式。CRISPR在菌株设计中的一个关键优势在于，它能够实现无标记DNA的染色体整合，省去了费力且往往效率低下的标记回收程序。尽管有这些优点，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工具包的一些主要局限性，并进行了改进设计，目的是使CRISPR技术更易于获取和实施。这些改进包括：1）一种使用表达Cre的菌株和标准菌株在无标记和基于标记的整合构建体之间快速切换的系统；2）通过基于金门的同源臂交换将多基因整合盒重定向到替代基因组位点的能力；3）一种通过Cas9辅助质粒和单链寡核苷酸之间的重组工程快速、简单地组装引导RNA序列的方法。我们将这些方法与成熟技术相结合，形成了一个使用CRISPR/Cas9进行高效代谢工程的综合工具包。作为概念验证，我们构建并表征了一个包含137个启动子的文库，并构建了一个能合成373.8mg/L尿黑酸的菌株。
