Tailoring a CRISPR/Cas9 cytidine base-editor enables fast and reliable construction of complex phenotypes in Pseudomonas species

Congreso; Congreso Conjunto SAIB-SAMIGE 2021; 2021

SAIB-SAMIGE

The genus Pseudomonas consists of species that inhabit a wide variety of environments, including soil and aquatic niches, in addition to plant and animal associations. The huge metabolic versatility of pseudomonads endowed them with the capacity of adapting to fluctuating environmental conditions, which is reflected in both their importance in plant and human disease as well as their expanding potential in biotechnological applications. Recent advances in genetic tools have improved our understanding of the Pseudomonas world. For instance, gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions, and substitutions have helped to link phenotypes to genotypes, and revealed novel genes function and molecular pathways. Yet, genetic manipulation methods in most Pseudomonas species remain time-consuming and tedious. The emergence of CRISPR/Cas9 and the development of base-editors opened innovative strategies for bacterial genome engineering. However, they are largely limited to one-by-one gene editions. In this work, we developed a multiplexed genetic tool specifically adapted to Pseudomonas sp. that speeds up the CRISPR-Cas9 gene-editing process. By harnessing the Csy4 endoribonuclease, a native component of the CRISPR locus of P. aeruginosa PA14, we placed multiple guide RNAs (gRNA) under the control of a single promoter, which is expressed as a single transcript and cleaved off by Csy4. The thereby generated multiple gRNA are in turn recognized by the Cas9 protein. Combining Golden Gate assembling and gRNA processing by Csy4, the method allows for up to 12 genes to be simultaneously edited. The windows of efficiency for the APOBEC1 deaminase into the 20-nucleotide spacer, from position 2 to 9 of the protospacer adjacent motif (PAM)-distal sequence, decreased as TC > AC > CC and dropped to nearly zero in GC edition events. The editing performance was enhanced by optimizing the incubation time, stabilizing the RNA motif, and by incorporating a uracil glycosylase inhibitor (UGI) to protect the guanine-uracil (G-U) intermediate. Combining all these features in a single, standard plasmid facilitated the creation of difficul-to-obtain mutants of P. putida and P. aeruginosa. Spacers in the multiplex gRNA displayed equal efficiency independently of their location and number, leading to a host-independent tool that can be used not only in Pseudomonas but also in other Gram-negative bacterial species.