RT Journal Article SR Electronic T1 Targetron-assisted delivery of exogenous DNA sequences into Pseudomonas putida through CRISPR-aided counterselection JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.05.01.442236 DO 10.1101/2021.05.01.442236 A1 Elena Velázquez A1 Yamal Al-Ramahi A1 Jonathan Tellechea A1 Natalio Krasnogor A1 Víctor de Lorenzo YR 2021 UL http://biorxiv.org/content/early/2021/05/01/2021.05.01.442236.abstract AB Genome editing methods based on Group II introns (known as Targetron technology) have been long used as a gene knock-out strategy in a wide range of organisms in a fashion independent of homologous recombination. Yet, their utility as delivery systems has been typically suboptimal because of their reduced efficiency of insertion when they carry exogenous sequences. We show that this limitation can be tackled and Targetron adapted as a general tool in Gram-negative bacteria. To this end, a set of broad host range standardized vectors were designed for conditional expression of the Ll.LtrB intron. After testing the correct functionality of these plasmids in Escherichia coli and Pseudomonas putida, we created a library of Ll.LtrB variants carrying cargo DNA sequences of different lengths to benchmark the capacity of intron-mediated delivery in these bacteria. Next, we combined CRISPR/Cas9-facilitated counterselection to increase the chances of finding genomic sites inserted with the thereby engineered introns. By following this pipeline, we were able to insert exogenous sequences of up to 600 bp at specific genomic locations in wild-type P. putida KT2440 and its ΔrecA derivative. Finally, we were able to apply this technology to successfully tag this strain with an orthogonal short sequence (barcode) that acts as a unique identifier for tracking this microorganism in biotechnological settings. The results with P. putida exemplified the value of the Targetron approach for unrestricted delivery of small DNA fragments to the genomes of Gram-negative bacteria for a suite of genome editing endeavours.Competing Interest StatementThe authors have declared no competing interest.