Repair of Double-Stranded DNA Breaks Generated by CRISPR–Cas9 in Pseudomonas putida KT2440

Abstract

Abstract Pseudomonas putida KT2440 is a metabolically versatile bacterium with considerable promise as a chassis strain for production and degradation of complex organic compounds. Unlike most bacteria, P. putida KT2440 encodes the Ku and LigD proteins involved in Non-Homologous End Joining (NHEJ). This pathway of repair of double-strand breaks (DSBs) in DNA has an intrinsic mutagenic potential that could be exploited in combination with currently available genome editing tools that generate programmable DSBs. Here, we investigated the effect of removal or overproduction of NHEJ-associated P. putida KT2440 enzymes on mutations generated upon repair of Cas9-mediated DSBs with the double purpose of characterizing the NHEJ pathway and investigating how it functionally interacts with the current gold standard tool for gene editing. The results of our work shed light on non-templated mechanisms of DSB repair in P. putida KT2440, an information that will serve as foundation to expand the gene engineering toolbox for this important microorganism.