Versioning Biological Cells for Trustworthy Cell Engineering

Tellechea‐Luzardo, Jonathan; L, Hobbs; Velázquez, Elena; Pelechova, Lenka; Woods, Simon; Lorenzo, de; Krasnogor, Natalio

doi:10.1101/2021.04.23.441106

Cited by 3 publications

(4 citation statements)

References 78 publications

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“…putida could benefit from a targeted, stable genomic insertion of short synthetic and orthogonal DNA sequences (i.e., genetic barcodes) as unique identifiers of particular strains. These barcodes create a physical link between the tagged organism and a digital twin, which in turn enables a version control system for microbial strains where all important information can be archived and consulted. , Given the broad host range and recombination-independent performance of the Ll.LtrB intron for directed insertion of small fragments of DNA, we evaluated its value for delivery of such barcodes/unique identifiers to the genomes of strains of interest. The barcodes of choice have a small size (148 bp, Supplementary Figure S4) and they are composed of a universal primer (25 nt), which is shared by all barcodes generated with CellRepo software, and a core sequence (123 nt).…”

Section: Results and Discussionmentioning

confidence: 99%

“…Next, we searched for adequate targeting loci in the genome of P. putida. As barcodes are meant to link a strain to its digital data, they need to be included in a stable and permissive genetic locus, 61,62 such as intergenic regions close to essential genes. The context close to glmS (close to the att Tn7 site, Figure 6) was thus selected as a good candidate for the insertion of Ll.LtrB::B3.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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

et al. 2021

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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 Δ 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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Section: Results and Discussionmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

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

et al. 2021

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“…Nonetheless, the optimization of the induction protocol as well as the insertion site should improve the efficiency of the insertion assay in any strain to be tested. In this way, we have succeeded in delivering small fragments (150 bp) into insertion loci even with low expected efficiency (low score) and a low‐efficiency spacer (90% cleavage efficiency) (Tellechea‐Luzardo et al., 2022; Velazquez et al., 2021).…”

Section: Commentarymentioning

confidence: 99%

“…The system can be employed to produce the same modification in different strains of the same species or to insert different cargo genes in one particular genomic site to generate diverse strains for testing. One interesting application could be the introduction of small synthetic pieces of DNA (i.e., genetic barcodes) for molecular labeling of different bacterial strains (Tellechea‐Luzardo et al., 2020, 2022). Since the main limitation of group II introns is the size of the insert that can be delivered (>600‐bp cargos can be difficult depending on the species), the insertion of small barcodes (∼150 bp) is a good example of the applicability of this system.…”

Section: Commentarymentioning

confidence: 99%

CRISPR/Cas9‐enhanced Targetron Insertion for Delivery of Heterologous Sequences into the Genome of Gram‐Negative Bacteria

2022

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Targetron technology, a gene‐editing approach based on the use of mobile group II introns, is particularly useful for bacterial strains deficient in homologous recombination. Specifically, the Ll.LtrB intron from Lactococcus lactis can be used in a wide range of species and can be easily retargeted, that is, modified for integration into any locus of interest. Targetron technology is thus a powerful tool for generating genomic insertions in a broad range of genetic backgrounds, mainly when no other techniques can be efficiently employed. Notably, the approach can be coupled to CRISPR/Cas9 counterselection of wildtype DNA sequences to decrease the population of unmodified cells and ultimately improve Ll.LtrB insertion efficiency. Here, we describe a step‐by‐step protocol for delivering exogenous sequences into the genome of Gram‐negative bacteria by means of targetron technology and CRISPR/Cas9 counterselection using Pseudomonas putida as a model. We describe the retargeting of the Ll.LtrB intron to the locus selected for insertion, the design of specific spacers for eliminating unmutated cells through CRISPR/Cas9 counterselection, and the cloning of exogenous sequences into Ll.LtrB. We also provide a protocol for delivering a specific cargo to the locus of choice once all necessary components of the system are ready. Lastly, we describe a general protocol for curing the engineered strain of all plasmids. CRISPR/Cas9‐enhanced Ll.LtrB insertion can be an efficient alternative for overcoming low recombination‐based editing efficiency and can be used in numerous bacterial species. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Retargeting the Ll.LtrB intron to the target locus Support Protocol 1: Preparation of competent E. coli Basic Protocol 2: Design and cloning of CRISPR spacers to counterselect Ll.LtrB insertions Support Protocol 2: Interference assay to check efficiency of selected spacers Basic Protocol 3: Cloning cargos into Ll.LtrB Basic Protocol 4: Ll.LtrB/CRISPR/Cas9‐mediated insertion Basic Protocol 5: Curing the engineered strain of plasmids

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Targetron-assisted delivery of exogenous DNA sequences intoPseudomonas putidathrough CRISPR-aided counterselection

Velázquez

Al‐Ramahi

Tellechea‐Luzardo

et al. 2021

Preprint

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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.

show abstract

Versioning Biological Cells for Trustworthy Cell Engineering

Cited by 3 publications

References 78 publications

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

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

CRISPR/Cas9‐enhanced Targetron Insertion for Delivery of Heterologous Sequences into the Genome of Gram‐Negative Bacteria

Targetron-assisted delivery of exogenous DNA sequences intoPseudomonas putidathrough CRISPR-aided counterselection

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