Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-asssisted, multiplex cytidine base-editing

SMANIA AM

NATURE COMMUNICATIONS

Nature

Año: 2022

2041-1723

RISPR-Cas technologies constitute a powerful tool for genome engineering, yet their use in non-traditional bacteria depends on host factors or exogenous recombinases that limit both efficiency and throughput. We have mitigated these practical constraints by developing a widely applicable genome engineering toolset for Gram-negative bacteria. The challenge was 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 supported multiplex base editing with >85% efficiency. The toolset was 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,