Modular design

Enzyme

Mathematics

Biology

Gene

Genome editing

Mathematical analysis

Programming language

Computer science

Computational biology

Cytidine

Multiplex

CRISPR

Base (topology)

Genetics

Biochemistry

Nature Communications

Rapid engineering of bacterial genomes is a requisite for both fundamental and applied studies. Here the authors develop an enhanced, broad-host-range cytidine base editor that enables multiplexed and efficient genome editing of Gram-negative bacteria.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 center dot G -> T center dot 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.

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