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.
Highlights
The accurate, site-specific genetic manipulation of mammalian chromosomes remains a technical challenge
As a general strategy to monitor site-specific donor integration in a preselected chromosomal target, the landing pad, we chose to follow the expression of two spectral distinct fluorescent proteins
The switch was from the landing pad vector (LPV)-encoded green fluorescent protein (GFP) to a red fluorescent protein (RFP)
Summary
The accurate, site-specific genetic manipulation of mammalian chromosomes remains a technical challenge. Site-specific targeting based on recombinases like Cre, Flp or Phi (see reference[2] for a recent review) has been used for decades without apparent cell type preferences While these recombinase systems are widely employed for cell line engineering, they inevitably require the prior establishment of master cell lines genetically engineered to contain suitably arranged recombinase recognition sites as chromosomal targets, referred to as “landing pads”. DNA double-strand cuts by these nucleases close to the intended insertion site facilitate a homology-driven repair (HDR) mechanism, provided that a properly designed DV that contains the insert flanked by homology arms identical to the chromosomal sequences around the actual cut site is present[16] Such alternative strategies, especially the use of RNA-guided engineered nucleases (RGENs) like in Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) genome editing, can substantially speed up targeted insertions when no premade master cell lines are available. When two potential integration sites were present in a given cell, striking differences between the recombinaseand the nuclease-based genome engineering systems were observed, with CRISPR/Cas[9] having a much higher capacity to simultaneously modify multiple loci at a time
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