Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, Delaware 19716, United States.
ACS Synth Biol. 2023 Oct 20;12(10):3082-3091. doi: 10.1021/acssynbio.3c00435. Epub 2023 Sep 28.
is an industrial host with a high fatty acid flux. Even though CRISPR-based tools have accelerated its metabolic engineering, there remains a need to develop tools for rapid multiplexed strain engineering to accelerate the design-build-test-learn cycle. Base editors have the potential to perform high-efficiency multiplexed gene editing because they do not depend upon double-stranded DNA breaks. Here, we identified that base editors are less toxic than CRISPR-Cas9 for multiplexed gene editing. We increased the editing efficiency by removing the extra nucleotides between tRNA and gRNA and increasing the base editor and gRNA copy number in a Ku70 deficient strain. We achieved five multiplexed gene editing in the ΔKu70 strain at 42% efficiency. Initially, we were unsuccessful at performing multiplexed base editing in NHEJ competent strain; however, we increased the editing efficiency by using a co-selection approach to enrich base editing events. Base editor-mediated canavanine gene (CAN1) knockout provided resistance to the import of canavanine, which enriched the base editing in other unrelated genetic loci. We performed multiplexed editing of up to three genes at 40% efficiency in the Po1f strain through the CAN1 co-selection approach. Finally, we demonstrated the application of multiplexed cytosine base editor for rapid multigene knockout to increase naringenin production by 2-fold from glucose or glycerol as a carbon source.
是一种具有高脂肪酸通量的工业宿主。尽管基于 CRISPR 的工具加速了其代谢工程，但仍需要开发用于快速多路复用电菌株工程的工具，以加速设计-构建-测试-学习周期。碱基编辑器有可能进行高效的多路基因编辑，因为它们不依赖双链 DNA 断裂。在这里，我们发现碱基编辑器比 CRISPR-Cas9 用于多路基因编辑的毒性更小。我们通过去除 tRNA 和 gRNA 之间的额外核苷酸，并在 Ku70 缺陷型菌株中增加碱基编辑器和 gRNA 的拷贝数，提高了编辑效率。我们在ΔKu70 菌株中以 42%的效率实现了五次多路基因编辑。最初，我们在非同源末端连接（NHEJ）能力的菌株中无法进行多路碱基编辑；然而，我们通过使用共选择方法来富集碱基编辑事件，从而提高了编辑效率。碱基编辑器介导的瓜氨酸基因（CAN1）敲除提供了对瓜氨酸导入的抗性，从而富集了其他不相关遗传基因座的碱基编辑。我们通过 CAN1 共选择方法在 Po1f 菌株中以 40%的效率进行了多达三个基因的多路编辑。最后，我们展示了多路胞嘧啶碱基编辑器在快速多基因敲除中的应用，以提高葡萄糖或甘油作为碳源时柚皮素的产量增加 2 倍。
