I’m happy to share my #PhD work on @biorxivpreprint. We used directed evolution to generate designer recombinases to correct a 140 kb genomic inversion causing Hemophilia A. 1/10
https://www.biorxiv.org/content/10.1101/2020.11.02.328013v1
Thank you! @BuchholzLab_TUD @TakebeLab @digsbb @tudresden_de @Medizin_TUD

Site-specific recombinases are powerful molecular genome editing tools and can perform various genomic modifications including inversions. Directed evolution allows us to redirect recombinases to target human genomic sites. 2/10

Severe Hemophilia A is often caused by large gene inversions between inverted repeats surrounding the F8 gene. We identified two asymmetric lox-like target sites (loxF8) in the int1h repeat regions as potential recombinase target sites. 3/10

Using two different evolution strategies revealed that we can generate either a single or a dual recombinase system that recombines the loxF8 sites in bacteria. The dual recombinase or heterodimer system was more active on the loxF8 target site. 4/10

Transfection of a HEK293T reporter cell line with mRNA coding for the loxF8 heterodimer showed robust recombinase activity in human cells. Additionally, a PCR-based assay revealed that the endogenous loxF8 locus was inverted. 5/10

The recombinase activity in human cells motivated us to investigate on potential off-target activity. Computationally predicted off-targets were tested in plasmid-based assay. Two of the tested off-targets were recombined albeit with lower efficiency as the on target. 6/10

Fusing the two monomers of the heterodimer improved the specificity and prevents homodimerization of the individual monomers. This might present a simple way to avoid activity of the monomers when using a heterodimer system. 7/10

We developed an experimental assay to identify potential binding sites of the fused heterodimer (RecF8). A ChIP-seq based experiment identified 85 binding sites. Twelve binding sites were tested in detail for recombination but no RecF8 activity was detected. 8/10

After expression of RecF8 in HEK293T we could show that 25-30% of the endogenous 140 kb loxF8 locus was inverted. A maximum of 50% inversion is possible using recombinases. We show for the first time that an evolved recombinase system can invert large genomic regions. 9/10

Treating endothelial cells differentiated from induced pluripotent stem cells of a hemophilic donor with RecF8 mRNA restored Factor VIII expression. This suggests a precise reversion, transcription and splicing of the F8 gene. 10/10