Dataset Information

Accelerated genome engineering of Pseudomonas putida by I-SceI-mediated recombination and CRISPR-Cas9 counterselection.

ABSTRACT: Pseudomonas species have become reliable platforms for bioproduction due to their capability to tolerate harsh conditions imposed by large-scale bioprocesses and their remarkable resistance to diverse physicochemical stresses. The last few years have brought forth a variety of synthetic biology tools for the genetic manipulation of pseudomonads, but most of them are either applicable only to obtain certain types of mutations, lack efficiency, or are not easily accessible to be used in different Pseudomonas species (e.g. natural isolates). In this work, we describe a versatile, robust and user-friendly procedure that facilitates virtually any kind of genomic manipulation in Pseudomonas species in 3-5 days. The protocol presented here is based on DNA recombination forced by double-stranded DNA cuts (through the activity of the I-SceI homing meganuclease from yeast) followed by highly efficient counterselection of mutants (aided by a synthetic CRISPR-Cas9 device). The individual parts of the genome engineering toolbox, tailored for knocking genes in and out, have been standardized to enable portability and easy exchange of functional gene modules as needed. The applicability of the procedure is illustrated both by eliminating selected genomic regions in the platform strain P. putida KT2440 (including difficult-to-delete genes) and by integrating different reporter genes (comprising novel variants of fluorescent proteins) into a defined landing site in the target chromosome.

SUBMITTER: Wirth NT

PROVIDER: S-EPMC6922521 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Accelerated genome engineering of Pseudomonas putida by I-SceI-mediated recombination and CRISPR-Cas9 counterselection.

Wirth Nicolas T NT Kozaeva Ekaterina E Nikel Pablo I PI

Microbial biotechnology 20190312 1

Pseudomonas species have become reliable platforms for bioproduction due to their capability to tolerate harsh conditions imposed by large-scale bioprocesses and their remarkable resistance to diverse physicochemical stresses. The last few years have brought forth a variety of synthetic biology tools for the genetic manipulation of pseudomonads, but most of them are either applicable only to obtain certain types of mutations, lack efficiency, or are not easily accessible to be used in different ...[more]

PMID: 30861315

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Project description:Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3'-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene ( neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.

Dataset Information

Accelerated genome engineering of Pseudomonas putida by I-SceI-mediated recombination and CRISPR-Cas9 counterselection.

Publications

Accelerated genome engineering of Pseudomonas putida by I-SceI-mediated recombination and CRISPR-Cas9 counterselection.

Similar Datasets

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