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.

Accelerating HIV‐1 VLP production using stable High Five insect cell pools

“…On top, different molecular biology approaches such as CRISPR/Cas9 [6] or recombinase-mediated cassette exchange (RMCE) [7] are used to this end since it is possible to direct the gene of interest (GOI) to genome loci with high transcription rates and low gene silencing. [8,9] Nevertheless, the development of efficient stable cell lines encompassing rapid cell growth rates, high cell concentrations, large recombinant protein yields, and product stability can be a very time-consuming and labor-intensive process that can take several months. [10] On the other hand, several studies have brought into debate the relevance of clonality by reporting the appearance of differentiated expression patterns during the culture of cells derived from a clonal cell line.…”

Nonmonotone invasion landscape by engineered control of metastasis activator levels

“…Thus, aiming to minimize genetic variability, we designed a two-step technical pipeline (Fig. 1B) to (i) introduce a FRT landing pad (LP) into AAVS1 upon CRISPR-Cas9 induced double strand breaks; and then (ii) flip-in various gene circuits into the LP by recombination without additional double strand breaks 70 .…”

On the Corner of Models and Cure: Gene Editing in Cystic Fibrosis

“…In this way, isogenic CFTR-variant cell lines of choice can be rapidly generated, all with an identical genetic background. While recombinases are commonly used to generate heterologous cell models, Cas9 in principle also supports cassette exchange although with slightly altered characteristics, such as a higher risk of knock-out alleles, or insertion of extra pieces of DNA alongside the integrated transgene ( Phan et al, 2017 ).…”