FLP Recombinase & Beta Resolvase Use Overview

What is FLP Recombinase?

In order to introduce the plasmid construct (BBa_K2605006) into the genome, utilising the CRISPR knock-in landing pad, FLP recombinase (FLP) was used. FLP binds to specific sites named FRT (flippase recombination target) sites. When FLP recognizes two target sites that have matching sequences, FLP binds, cleaving both sites allowing for a “crossing over” event to take place in which FLP ligates the two sequences together. While the exact mechanism is hard to visualise at the nucleotide level, it is simply understood by comparing the before and after of the recombination event.

In our system an FRT site is found within the genome of our modified HEK293T cells and another, complimentary, FRT site is located on the plasmid construct (BBa_K2605006), along with another plasmid (pCAG-FLPo) containing the gene coding for FLP. Once the plasmids are introduced to the HEK293T cells pCAG-FLPo transiently expresses FLP, which then recognizes the complimentary FRT sites. When FLP cleaves and ligates these sites, the entire plasmid construct is integrated into the genome, thus the genome now contains all genetic elements from both the CRISPR knock-in and the plasmid construct.

This recombination is supplemented by the 𝛽-resolvase (secondary recombination event), which removes the possibility of FLP naturally excising the inserted plasmid construct through the reverse reaction, which is favorable.

What is Beta Resolvase?

Integration of Hybrid FLPo - Beta Resolvase Into HEK293T Genome

Design of Dual Recombinase Approach

Recombinase Mediated Cassette Exchange

The use of recombinase mediated cassette exchange (RMCE) was initially our chosen method for genomic integration, however it was replaced by the dual recombinase approach due to the inherent issue of the natural removal of the insert from the genome by FLP. RMCE utilises two pairs of FRT sites in which they flank a cassette in opposing directions.

With this method, the landing pad, inserted using the CRISPR knock-in, would have been larger in size, thus reducing efficiency. This FRT pair construct would have been mirrored on our plasmid construct, in which FLP activity would quite literally flip the cassette (contained between the FRT pair) from in between the plasmid FRT sites and the genomic FRT sites through crossing over events between matching FRT sites. The issue with RMCE lies in the equilibrium reaction that favours the removal of the larger cassette from the genome, making it infeasible to utilise for our purposes.