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Schmitt, L. T.

Publications and source records attributed to Schmitt, L. T..

2 recordsLinked to original sources

Prediction of designer-recombinases for DNA editing with generative deep learning

Site-specific tyrosine-type recombinases are effective tools for genome engineering, with the first engineered variants having demonstrated therapeutic potential. So far, adaptation to new DNA target site selectivity of designer-recombinases has been achieved mostly through iterative cycles of directed molecular evolution. While effective, directed molecular evolution methods are laborious and time consuming. Here we present RecGen (Recombinase Generator), an algorithm for the intelligent generation of designer-recombinases. We gathered the sequence information of over two million Cre-like recombinase sequences evolved for 89 different target sites with which we trained Conditional Variational Autoencoders for recombinase generation. Experimental validation demonstrated that the algorithm can predict recombinase sequences with activity on novel target-sites, indicating that RecGen is useful to accelerate the development of future designer-recombinases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/486669v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@c08326org.highwire.dtl.DTLVardef@e26984org.highwire.dtl.DTLVardef@7d7542org.highwire.dtl.DTLVardef@9d6642_HPS_FORMAT_FIGEXP M_FIG C_FIG Teaser Figure: Recombinase prediction generates active recombinases for a desired target site, while existing libraries with a similar target site need to go through directed evolution to achieve activity on the new site. While evolution takes weeks, prediction and synthesis of recombinases can be done in days.

bioinformatics↗

Correction of a Factor VIII genomic inversion with designer-recombinases

Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct a 140 kb genomic inversion of the F8 gene, which is frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we developed a fused heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, resulted in prominent correction of the inversion and restored Factor VIII mRNA expression. Our data suggests that designer-recombinases may represent efficient and specific means towards treatment of monogenic diseases caused by large gene inversions.

molecular biology↗