Dataset Information

Site-specific chromosomal gene insertion: Flp recombinase versus Cas9 nuclease.

ABSTRACT: Site-specific recombination systems like those based on the Flp recombinase proved themselves as efficient tools for cell line engineering. The recent emergence of designer nucleases, especially RNA guided endonucleases like Cas9, has considerably broadened the available toolbox for applications like targeted transgene insertions. Here we established a recombinase-mediated cassette exchange (RMCE) protocol for the fast and effective, drug-free isolation of recombinant cells. Distinct fluorescent protein patterns identified the recombination status of individual cells. In derivatives of a CHO master cell line the expression of the introduced transgene of interest could be dramatically increased almost 20-fold by subsequent deletion of the fluorescent protein gene that provided the initial isolation principle. The same master cell line was employed in a comparative analysis using CRISPR/Cas9 for transgene integration in identical loci. Even though the overall targeting efficacy was comparable, multi-loci targeting was considerably more effective for Cas9-mediated transgene insertion when compared to RMCE. While Cas9 is inherently more flexible, our results also alert to the risk of aberrant recombination events around the cut site. Together, this study points at the individual strengths in performance of both systems and provides guidance for their appropriate use.

SUBMITTER: Phan QV

PROVIDER: S-EPMC5736728 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Site-specific chromosomal gene insertion: Flp recombinase versus Cas9 nuclease.

Phan Quang Vinh QV Contzen Jörg J Seemann Petra P Gossen Manfred M

Scientific reports 20171219 1

Site-specific recombination systems like those based on the Flp recombinase proved themselves as efficient tools for cell line engineering. The recent emergence of designer nucleases, especially RNA guided endonucleases like Cas9, has considerably broadened the available toolbox for applications like targeted transgene insertions. Here we established a recombinase-mediated cassette exchange (RMCE) protocol for the fast and effective, drug-free isolation of recombinant cells. Distinct fluorescent ...[more]

PMID: 29259215

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Project description:Pseudomonas sp. strain B13 carries the clcRABDE genes encoding chlorocatechol-degradative enzymes on the self-transmissible 105-kb clc element. The element integrates site and orientation specifically into the chromosomes of various bacterial recipients, with a glycine tRNA structural gene (glyV) as the integration site. We report here the localization and nucleotide sequence of the integrase gene and the activity of the integrase gene product in mediating site-specific integration. The integrase gene (int-B13) was located near the right end of the clc element. It consisted of an open reading frame (ORF) of maximally 1,971 bp with a coding capacity for 657 amino acids (aa). The full-length protein (74 kDa) was observed upon overexpression and sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation. The N-terminal 430 aa of the predicted Int-B13 protein had substantial similarity to integrases from bacteriophages of the P4 family, but Int-B13 was much larger than P4-type integrases. The C-terminal 220 aa of Int-B13 were homologous to an ORF flanking a gene cluster for naphthalene degradation in Pseudomonas aeruginosa PaK1. Similar to the bacteriophages phiR73 and P4, the clc element integrates into the 3' end of the target tRNA gene. This target site was characterized from four different recipient strains into which the clc element integrated, showing sequence specificity of the integration. In Pseudomonas sp. strain B13, a circular form of the clc element, which carries an 18-bp DNA sequence identical to the 3'-end portion of glyV as part of its attachment site (attP), could be detected. Upon chromosomal integration of the clc element into a bacterial attachment site (attB), a functional glyV was reconstructed at the right end of the element. The integration process could be demonstrated in RecA-deficient Escherichia coli with two recombinant plasmids, one carrying the int-B13 gene and the attP site and the other carrying the attB site of Pseudomonas putida F1.

Dataset Information

Site-specific chromosomal gene insertion: Flp recombinase versus Cas9 nuclease.

Publications

Site-specific chromosomal gene insertion: Flp recombinase versus Cas9 nuclease.

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