Adenine Base Editing System for Pseudomonas and Prediction Workflow for Protein Dysfunction via ABE

Pseudomonas is a large genus that inhabits diverse environments due to its distinct metabolic versatility. Its applications range from environmental to industrial biotechnology. Molecular tools that allow precise and efficient genetic manipulation are required to understand and harness its full potential. Here, we report the development of a highly efficient adenine base editing system, i.e., dxABE-PS, for Pseudomonas species. The system allows A:T -> G:C transition with up to 100% efficiency along a broad target spectrum because we use xCas9 3.7, which recognizes NG PAM. To enhance the cbcABE-PS utility, we develop a prediction workflow for protein dysfunction using ABE, namely, DARE-CSP (dysfunction via ABE through CRISPOR-SIFT prediction). We applied DABE-CSP to inactivate several genes in Pseudomonas putida KT2440 to accumulate a nylon precursor, i.e., muconic acid from catechol with 10096 yield. Moreover, we expanded the ABE to non-model Pseudomonas species by developing an nxABE system for P. chengduensisDY56-96, isolated from sediment samples from the seamount area in the West Pacific Ocean. Taken together, the establishment of the ABE systems along with DABE-CSP will fast-track research on Pseudomonas species.

Automated summary

This study reported the development of a highly efficient adenine base editing system for Pseudomonas species, providing molecular tools for understanding and harnessing the potential of this genus. The study also developed a prediction workflow for protein dysfunction, expanding the application of the editing system to non-model Pseudomonas species.