Novel Recombinases and Target Sequences

Technology #17809

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Conserved motifs within the catalytic domain and dimerization helix (αE) of large serine recombinases. Motifs A and C contain the critical active site residues of the recombinase. Motif D, contained within the C-terminal portion of the E-helix plus a few residues beyond, is mostly involved with binding the DNA in the region abutting the cleavage site. Motif B forms a rather mobile loop whose function remains mysterious despite the remarkable conservation of the Ser-39, Gly-40, and Arg-45 residues.Phylogenic tree of identified large serine recombinase sequences. The 26 recombinases together with Peaches and BxZ2. The scale bar indicates a difference of 2 amino acids.
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Inventors
Professor Ron Weiss
Department of Biological Engineering, MIT
External Link (groups.csail.mit.edu)
Yinqing Li
Department of Electrical Engineering & Computer Science, MIT
Xavier Duportet
Department of Electrical Engineering & Computer Science, MIT
Gregory Batt
inria
Managed By
Jon Gilbert
MIT Technology Licensing Officer
Patent Protection

Novel Recombinases and Target Sequences

US Patent Pending

Novel Recombinases and Target Sequences

PCT Patent Application Filed

Applications

This invention is a method to identify serine recombinases for the manipulation of the mammalian genome. There are a number of specific applications including: incorporating in an inducible positive or negative feedback loop allowing the monitoring and modeling of mammalian cellular behavior or the integration of peptides, RNAs and proteins for enhanced controlled or continuous therapeutic expression.

Problem Addressed

Programming mammalian cells with large synthetic gene networks can significantly facilitate elucidating complex regulatory cellular mechanisms, implementing new useful biological functions and accelerating the design of novel tailor-made therapeutic treatments. Complex, stable and heritable programming of mammalian cells by genomic engineering is limited by the requirement to pre-integrate a natural recombination site at single or multiple genomic (chromosomal) loci, thus necessitating the identification of programmable orthogonal (independently acting) recombinases that can be directly targeted sequentially or simultaneously to the endogenous sequences of choice in the mammalian genome. However, only a handful of these enzymes have yet been discovered and are extremely hard to engineer given the complexity of the site‐specific recombination mechanism. This technology includes the discovery of new large serine recombinases, shown to be the most promising ones in terms of efficiency and specificity for the manipulation of mammalian genomes, and procedures to identify more serine recombinases.

Technology

This technology includes new large serine recombinases identified from recently sequenced mycobacteriophage genomes and a dedicated plasmid rescue system to identify the specific attB/attP recombination sites recognized by these recombinases. The ability of multiplex integration by using orthogonal sites could help to integrate a variety of different sequences, including circuits containing more than one sequence each, at different locations within a genome, which is helpful in preventing interference between circuits or attaining higher levels of expression. The inventors created a script to scan phage genomes and identify putative large serine recombinases as well as a suite of plasmids to enable the discovery of their recombination sites in their natural host and to transpose the system in E. coli and mammalian cells. The system was validated by identifying 26 new large serine recombinases from 400 Mycobacteriophage genomes, from which 4 were using new recombination sites.

Advantages

  • Identify serine recombinases for varied uses in synthetic biology
  • Can integrate in a variety of sequences at different locations in the genome