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

Lookup NU author(s): Jonathan Tellechea Luzardo, Professor Natalio KrasnogorORCiD

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Abstract

© 2021 The Authors. Published by American Chemical Society. 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.


Publication metadata

Author(s): Velazquez E, Al-Ramahi Y, Tellechea-Luzardo J, Krasnogor N, De Lorenzo V

Publication type: Article

Publication status: Published

Journal: ACS Synthetic Biology

Year: 2021

Volume: 10

Issue: 10

Pages: 2552-2565

Print publication date: 15/10/2021

Online publication date: 02/10/2021

Acceptance date: 03/05/2021

Date deposited: 09/11/2023

ISSN (electronic): 2161-5063

Publisher: American Chemical Society

URL: https://doi.org/10.1021/acssynbio.1c00199

DOI: 10.1021/acssynbio.1c00199


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Funding

Funder referenceFunder name
BioRoboost
Comunidad de Madrid
ERA-COBIOTECH 2018—PCI2019-111859-2
Engineering and Physical Sciences Research Council (EPSRC)
EP/N031962/1EPSRC
European Structural and Investment Funds
European Union
InGEMICS-CM
FSE, FECER
H2020-FET-OPEN-RIA-2017-1-766975
H2020-NMBP-BIO-CSA-2018-820699
H2020-NMBP-TR-IND/H2020-NMBP-BIO-2018-814650
MINECO/FEDER
MIX-UP H2020-BIO-CN-2019-870294
MADONNA
MIX-UP
S2017/BMD-3691
SyCoLiM
SynBio4Flav
Royal Academy of Engineering Chair in Emerging Technologies
RTI2018-095584-B-C42
SETH
Spanish Ministry of Science and Innovation

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