4.7 Article

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

Journal

ACS SYNTHETIC BIOLOGY
Volume 10, Issue 10, Pages 2552-2565

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.1c00199

Keywords

Pseudomonas putida; targetron; genome editing; CRISPR/Cas9; barcode; orthogonal DNA

Funding

  1. SETH Project of the Spanish Ministry of Science and Innovation [RTI2018-095584-B-C42]
  2. SyCoLiM Project of the Spanish Ministry of Science and Innovation [ERA-COBIOTECH 2018-PCI2019-111859-2]
  3. European Union [H2020-FET-OPEN-RIA-2017-1-766975, H2020-NMBP-BIO-CSA-2018-820699, H2020-NMBP-TR-IND/H2020-NMBP-BIO-2018-814650, MIX-UP H2020BIO-CN-2019-870294]
  4. InGEMICS-CM Project of the Comunidad de Madrid-European Structural and Investment Funds-(FSE, FECER) [S2017/BMD-3691]
  5. Education Ministry, Madrid, Spanish Government [FPU15/04315]
  6. Engineering and Physical Sciences Research Council (EPSRC) [EP/N031962/1]
  7. Royal Academy of Engineering Chair in Emerging Technologies award
  8. EPSRC [EP/N031962/1] Funding Source: UKRI

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Research demonstrates that by improving targetron technology, exogenous sequences can be successfully inserted into the genomes of Gram-negative bacteria. Through the establishment of a series of standardized vectors and the combination of CRISPR/Cas9-facilitated counterselection, successful intron-mediated DNA delivery in bacteria was achieved, and P. putida microorganism was successfully tagged.
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 Delta recA 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.

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