The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.
Departamento de Química Biológica Ranwel Caputto, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.
Nat Commun. 2022 May 31;13(1):3026. doi: 10.1038/s41467-022-30780-z.
CRISPR/Cas technologies constitute a powerful tool for genome engineering, yet their use in non-traditional bacteria depends on host factors or exogenous recombinases, which limits both efficiency and throughput. Here we mitigate these practical constraints by developing a widely-applicable genome engineering toolset for Gram-negative bacteria. The challenge is addressed by tailoring a CRISPR base editor that enables single-nucleotide resolution manipulations (C·G → T·A) with >90% efficiency. Furthermore, incorporating Cas6-mediated processing of guide RNAs in a streamlined protocol for plasmid assembly supports multiplex base editing with >85% efficiency. The toolset is adopted to construct and deconstruct complex phenotypes in the soil bacterium Pseudomonas putida. Single-step engineering of an aromatic-compound production phenotype and multi-step deconstruction of the intricate redox metabolism illustrate the versatility of multiplex base editing afforded by our toolbox. Hence, this approach overcomes typical limitations of previous technologies and empowers engineering programs in Gram-negative bacteria that were out of reach thus far.
CRISPR/Cas 技术是基因组工程的强大工具，但它们在非传统细菌中的应用取决于宿主因素或外源重组酶，这限制了效率和通量。在这里，我们通过开发一种广泛适用于革兰氏阴性菌的基因组工程工具集来减轻这些实际限制。通过定制一种 CRISPR 碱基编辑器来解决这一挑战，该编辑器能够实现单核苷酸分辨率的操作（C·G→T·A），效率超过 90%。此外，在简化的质粒组装方案中整合 Cas6 介导的向导 RNA 加工，支持超过 85%效率的多重碱基编辑。该工具集用于构建和分解土壤细菌恶臭假单胞菌中的复杂表型。芳香化合物生产表型的单步工程和复杂氧化还原代谢的多步分解说明了我们工具盒提供的多重碱基编辑的多功能性。因此，这种方法克服了以前技术的典型限制，并为革兰氏阴性菌的工程计划提供了支持，这些计划迄今为止是遥不可及的。
