Targetron-Assisted Delivery of Exogenous DNA Sequences into Pseudomonas putida through CRISPR-Aided Counterselection.

ABSTRACT: Genome editing methods based on group II introns (known as targetron technology) have long been used as a gene knockout strategy in a wide range of organisms, in a fashion independent of homologous recombination. Yet, their utility as delivery systems has typically been suboptimal due to the reduced efficiency of insertion when carrying exogenous sequences. We show that this limitation can be tackled and targetrons can be adapted as a general tool in Gram-negative bacteria. To this end, a set of broad-host-range standardized vectors were designed for the conditional expression of the Ll.LtrB intron. After establishing 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

SUBMITTER: Velazquez E 

PROVIDER: S-EPMC8524655 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature