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An extended DNA recombinase toolkit for mammalian systems

Identifying, screening and optimising a novel recombinase toolkit for mammalian cells.

The Idea

DNA recombinases perform excision, integration and inversion events on recognition of pairs of cognate DNA recombinase recognition sites. The outcome of the recombination event is directed by the orientation of the two recognition sites. These functions make valuable tools for a wide variety of applications including DNA assembly, control of gene expression, mutation, information processing (e.g. logic gates) and gene delivery/therapy. The field of synthetic biology has used these enzymes to build a variety of interesting devices, including memory modules, cellular counters and the full gamut of logic gates. However, the more sophisticated recombinase-based devices have so far been limited to use in prokaryotic organisms. This is in part due to the limited number of recombinases with high efficiency in other (e.g. mammalian) systems. 


We are particularly interested in the application of recombinases to investigating the neural circuits that subserve specific brain functions and associated disorders. A principal means of investigating this is to express reporter or effector genes in specific pathways at specific times either by delivery of synthetic genetic constructs via viruses or by generating transgenic animal models. This relies on a genetic reporter construct containing recombinase recognition sites designed such that reporter gene expression occurs only in the presence ofrecombinase activity (e.g. excision or inversion). The combination of injection of a retrograde virus carrying a recombinase expression cassette into brain structure A and injection of a second virus containing the reporter construct into brain structure B results in specific labelling of neurons in structure B that project to A. This technique has yielded interesting data on neuroanatomy. However the complexity of brain networks is such that structures typically receive inputs from several other loci. In these circumstances, multiple recombinases are required in order to simultaneously map these multiple circuits and their associated function. Moreover, the efficiency of theserecombinases will become increasingly important where outputs depend on the spatial intersection of their activity. 

We are currently limited in our designs by the small number of high-activity recombinases available. There are currently just two tyrosinerecombinase - Cre and Flp - with high activity across common cell types. Many recombinases have been identified, for example in microbial and phage genomes, however, their activity in mammallian cells is likely to be poor without sequence optimisation. We therefore propose to identify, screen and optimise (e.g. GC content, codon usage) a set recombinases whose activity in mammalian cells have not yet been reported. 


We would propose to: 
1) Identify candidate recombinases (principally from literature) 
2) Optimise the DNA sequences of the recombinsases in silico (GC content, codon usage etc). 
3) Have the optimised DNA sequences synthesised and clone into a repoter construct. 
4) Test the activity and orthogonality (i.e. test for specific for each recombinase for its cognate target sequence vs those of otherrecombinases) in fibroblasts using a transient transfection assay. 
5) Test the most promising recombinases in other mammalian cell lines. 

The recombinase activity data would be made public through publication online and the physical DNA sequences would be made available via Addgene. 

We think the availability of a larger set of high-efficiency recombinases would extend the utility of these versatile tools to a new range of varied applications including those application mentioned. The proposal also has the potential to benefit applications in eukaryotic systems beyond mammals, since the process of optimisation of recombinases from prokaryotic systems would likely involve similar hurdles (e.g. GC content). 

 

The Team

Dr Peter Davenport
Maxime Fouyssac
Generic Avatar
Haydn King

 

 

 

 

 

 

 

Peter Davenport

Research Assistant, Department of Pathology

Maxime Fouyssac

PhD student, Department of Pharmacology

Haydn King 

Graduate student, Department of Pathology

 

Project Outputs

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The Synthetic Biology Strategic Research Initiative provides a hub for anyone interested in Synthetic Biology at the University of Cambridge, including researchers, commercial partners and external collaborators. 

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