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 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. With these novel tools, 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 applied this technology to successfully tag P. putida with an orthogonal short sequence barcode that acts as a unique identifier for tracking this microorganism in biotechnological settings. These results show the value of the targetron approach for the unrestricted delivery of small DNA fragments to precise locations in the genomes of Gram-negative bacteria, which will be useful for a suite of genome editing endeavors.
Highlights
Genome editing methods based on group II introns have long been used as a gene knockout strategy in a wide range of organisms, in a fashion independent of homologous recombination
ACS Synthetic Biology pubs.acs.org/synthbio founded on the Ll.LtrB group II intron from Lactococcus lactis since it is the most studied case and it was proven to work in a wide range of bacterial genera, from Clostridium[24] or Bacillus[25] to the well-characterized species Escherichia coli.[23]
Since a retrotransposition-activation selectable marker (RAM) is placed inside Ll.LtrB, kanamycin resistance was used as a way to select for intron insertion mutants. (B) Graph shows the number of KmR CFU normalized to 109 viable cells and classified according to the displayed phenotype in the presence of X-gal (blue colonies: lacZ+, blue bars; white colonies: lacZ−, white hatched bars) and, according to the presence or absence of IPTG induction. (C)
Summary
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. ACS Synthetic Biology pubs.acs.org/synthbio founded on the Ll.LtrB group II intron from Lactococcus lactis since it is the most studied case and it was proven to work in a wide range of bacterial genera, from Clostridium[24] or Bacillus[25] to the well-characterized species Escherichia coli.[23] Later, targetrons were assayed for the delivery of specific cargo sequences into designated loci.[26−28] Group II introns are promising tools to this end as they give rise to stable integrations.[26,28] Another useful feature is that they are broadhost-range actors that can work in a great variety of organisms.[24,25,27,29,30] they can be redirected to virtually any desired genomic location with high specificity.[23,31] as already mentioned, they can be an alternative to homologous recombination-based techniques, as they function independently of recA, which is a considerable advantage compared to other editing systems.[32,33]. Since a RAM is placed inside Ll.LtrB, kanamycin resistance was used as a way to select for intron insertion mutants (plates to the right). (B) Graph shows the number of KmR CFU normalized to 109 viable cells and classified according to the displayed phenotype in the presence of X-gal (blue colonies: lacZ+, blue bars; white colonies: lacZ− (disrupted), white hatched bars) and, according to the presence or absence of IPTG induction. (C)
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