Identifying, screening and optimising a novel recombinase toolkit for mammalian cells. Full document.
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 [1,2]. 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 of recombinase activity (e.g. excision or inversion, [3]). 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 these recombinases 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 tyrosine recombinase - Cre and Flp - with high activity across common cell types. Many recombinases have been identified, for example in microbial and phage genomes (e.g. [4]), 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 other recombinases) 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).
[1] Friedland AE, Lu TK, Wang X, Shi D, Church G, Collins JJ. 2009. Synthetic Gene Networks That Count. Science 324:1199–1202.
[2] Siuti P, Yazbek J, Lu TK. 2013. Synthetic circuits integrating logic and memory in living cells. Nat Biotech 31:448–452.
[3] Denny CA, Kheirbek MA, Alba EL, Tanaka KF, Brachman RA, Laughman KB, Tomm NK, Turi GF, Losonczy A, Hen R. 2014. Hippocampal Memory Traces Are Differentially Modulated by Experience, Time, and Adult Neurogenesis. Neuron 83:189–201.
[4] Yang L, Nielsen AAK, Fernandez-Rodriguez J, McClune CJ, Laub MT, Lu TK, Voigt CA. 2014. Permanent genetic memory with >1-byte capacity. Nat Meth 11:1261–1266.
Who we are:
Peter Davenport (pwd22) RA, Ajioka Group, Department of Pathology. Currently developing foundational tools in Synthetic Biology as part of the FLOWERS synthetic biology consortium. Undergraduate and master degrees in Natural Sciences (Biochemistry, Cambridge University). PhD research in Biochemistry and Microbiology (Department of Biochemistry, Cambridge University).
Maxime Fouyssac (mf539) PhD student in the Belin laboratory of “Neuropsychopharmacology of Compulsive Disorders”, Department of Pharmacology. Maxime Fouyssac has developed primary cultures of astrocytes from micro dissected rat brains, capitalising on his previous expertise in cell culture. As part of his PhD research project, Maxime Fouyssac aims to causally manipulate the expression of candidate genes in striatal astrocytes alongside ontogenetic manipulation of adjacent selective neuronal populations. The development of new recombinases would be of great benefit to application of this type. He will benefit from the full support of his supervisor and will have access to the necessary equipment.
Haydn King (hjk38) First year PhD student in synthetic biology the Akioka lab. Studied Engineering at Cambridge as an undergraduate - specialising in systems and signal processing - before completing an MRes in biological sciences prior to beginning the PhD.
Implementation:
Implementation Aim Develop recombinases with high activity in mammalian cells. Work package 1 Selection of candidate phage integrases Identify a set of ~10 candidate recombinases, principally from the literature, although a sequence based bioinformatic search will also be conducted. The phage integrases identified by Yang et al. [1] would be a major starting point. Criteria for selection will include "distance to optimisation" (i.e. to what extent will the sequence need modifying) and orthogonality of recognition sites. [PWD, HK, MF] Optimisation of recombinases for expression in mammalian cells. This will include consideration of codon optimisation, optimisation of GC content, mRNA structure. [PWD, HK, MF] Vector construction. Several strategies have been used to assay recombinase activity. We favour use of a plasmid born inversion-detector. Recombinase activity would result in inversion and hence expression of a fluorescent protein ORF flanked by recombinase recognition sites. "Reference" fluorescent reporter genes will be used to control for transfection efficiency. [PWD] Synthesis. We will outsource DNA synthesis of optimised recombinase sequences to a commercial DNA synthesis company. Candidate companies include Gen9bio, GeneArt and IDT. (Currently ~£250 for 1.5-2 kbp.) [PWD] Work package 2 Testing in Mammalian cell culture Fibroblast (3T3) cell cultures will be transiently transfected with the recombinase and reporter vectors. Fluorescence reporter signals will be measured in via fluorescence microscopy. We will also investigate whether use of a fluorescence microtitre plate reader (BMG) can be used to increase throughput. Transfection efficiency will be assayed using the reference reporters. The recombinase activity will be assayed using the ratio of signals from the inversion-detector and the reference reporters. [MF, PWD] Work package 3 Recombinases with high activity (e.g. ~>80%) will be considered for testing in primary neuronal cell culture (as a proof of concept for live animal studies). [MF] Work package 4 (followon) Depending on the success of the above work, we would like to assess activity in vivo in the rat brain. This will be important in assessing whether the recombinase activities are sufficient for the specific purpose outlined previously. This would involve AAV encapsulation of the reporter and recombinase constructs followed by delivery to specific structures (centering on the nucleus accumbens) in the rat brain. [1] Yang L, Nielsen AAK, Fernandez-Rodriguez J, McClune CJ, Laub MT, Lu TK, Voigt CA. 2014. Permanent genetic memory with >1-byte capacity. Nat Meth 11:1261–1266.
Benefits and outcomes:
The direct outcomes of the work in this proposal would be a new set of DNA recombinases optimised and characterised for activity in mammalian cells. As mentioned above, there are huge variety of potential applications beyond those specifically described here, including gene delivery and information processing (e.g. logical gates). The recombinases developed in this work are also likely to be of use to researchers working in other eukaryotic systems. The characterisation and sequence data will be published online and the physical DNA made available via Addgene. These resources would be a valuable contribution to the open technology base surrounding DNA recombinases and would facilitate researchers to rapidly adapt these tools to their own ends. This proposal would initiate a new interdisciplinary collaboration between researchers currently working on foundational Synthetic Biology tools (principally for use in biosenors) with behavioural neuroscientists (with a particular interest in addiction). We hope that this collaboration will spark further discussion and collaboration beyond the immediate subject of this proposal. The proposal has been carefully designed to be achievable within the time period allocated and members of the group have all necessary expertise to proceed successfully, including expertise in the relevant bioinformatic, molecular biology and cell culture techniques. We hope this work will be a basis for the sharing of such expertise amongst its participants.
Budget:
Gene synthesis £250 x 10 genes = £2,500 Molecular biology reagents £300 Cell culture reagents £1200