The Role of NHEJ Double-Strand Break Repair on CRISPR-Cas9 Targeting in Pseudomonas Putida KT2440

A novel three-plasmid system of targeted genomic engineering in Pseudomonas putida KT2440 has been developed in our laboratory. It includes ssDNA recombineering followed by Cas9 counterselection¹. So far, point mutations and deletions have been performed by our method, however the efficiency of counterselection has proven to be low in the majority of targeted loci. In contrast to most prokaryotes, P. putida possesses NHEJ associated genes, encoding for Ku and LigD proteins, whose activity has been associated with stationary-phase mutagenesis in starving P. putida².

In this work we (1) investigated whether the low counterselection efficiency is caused by a non-templated error prone repair of the double-stranded break (DSB) performed by Cas9-sgRNA efector and (2) evaluated the posible role of Ku and LigD proteins in the process.

We found that, unlike many other bacteria, P. putida can overcome the Cas9-associated toxicity by repairing the DSB in error prone way. Even more, knocking/overexpressing ku or ligD genes did not affect the counterselection efficiency but did change the mutation spectrum in the target region, suggesting that, besides the canonical NHEJ, other alternative pathways may be involved in the repair event.

The understanding of how DSBs are repaired via NHEJ in P. putida will contribute to the optimization of editing strategies based on CRISPR/Cas9 counterselection.

[1] Quintana Cordero J. ssDNA recombineering followed by Cas9/sgRNA counterselection as a new tool for Pseudomonas putida genome editing. Master’s Thesis, Skolkovo Institute of Science and Technology, 2018.

[2] Paris U., Mikkel K., Tavita K., Saumaa S., Teras R., Kivisaar M. (2015) NHEJ enzymes LigD and Ku participate in stationary-phase mutagenesis in Pseudomonas putida. DNA Repair 31, 11-18.