New recombinases for genome engineering

Site-specific recombinases are a class of proteins that splice different DNA sequences together in a specific and predictable way. They are widely used in biotechnology to manipulate the genomes of plants and animals. A particularly useful application of these proteins is to insert or delete genes or segments of DNA in mammalian chromosomes to study their function. Ideally inserted DNA should be prevented from excising (and excised DNA must be prevented from re-inserting) and this can be done with one group of site-specific recombinases called the serine integrases. One of us, Prof Smith, discovered that the integrase from a bacterial virus, phiC31 can promote an irreversible insertion or deletion without the participation of any other protein. This protein has, as a consequence, become very widely used in nearly all branches of experimental biology. phiC31 integrase is not perfect however and in fact suffers from major problems. Sometimes the reaction promoted by phiC31 integrase is incomplete and sometimes it does not happen at all. Many other serine recombinases have been discovered in the course of genome projects and some may be much better than the original phiC31 integrase for use in biotechnology. Surprisingly these new proteins have never been studied systematically in order to determine whether any of them are in fact better than phiC31 integrase. We would like to do this.

The Streptomyces phiC31 bacteriophage integrase is a unidirectional site-specific recombinase that is widely used in genome engineering. However phiC31 integrase is not ideal as a genome engineering reagent because it suffers the following problems: 1. Recombination sites are frequently destroyed by small deletions. We propose this is because an intermediate in the recombination reaction can be recognized as damaged DNA and is repaired by cellular proteins before integrase can complete its reaction. As a result 20%-50% of the reactions may fail depending on the experimental conditions. This renders phiC31 integrase unsuitable for a whole set of important manipulations. 2. The rate or efficiency of the site-specific recombination reaction is liable to chromosomal position effects in eukaryotic cells. 3. It is currently hard to engineer a conditionally active version of the phiC31 integrase 4. Some cell types require two recombination sites on both the target and incoming DNA, presumably due to low occupancy when only a single site is present. 5. The native protein is encoded by a gene with the codon usage characteristic of Streptomyces and is poorly expressed in eukaryotic cells. Many new integrases have been discovered in the course of genome projects and some may be better than phiC31 integrase for genome engineering in one or all of these respects. We will compare fourteen integrases for which the attP and attB sites are known in a series of chromosome engineering reactions in cell culture in order to identify if they vary in their predisposition to these problems and which might suit specific purposes better than phiC31 integrase. At the same time we will study the novel integrase/att site systems in vitro to ask how much the eukaryotic cell environment affects integrase activity and to understand the bases for the problems with phiC31 integrase. This research will lead to improved reagents for genome engineering and genetic modification.

Our research will benefit those who make genetic modifications to cells, animals, plants and microbes because it will give them an improved set of tools and a better understanding of any factors that may limit the utility of these tools. We will ensure dissemination of these benefits by forming a UK genome engineering club and by establishing annual genome engineering workshops. Genome modification is a fundamental technique employed across the whole of the biotechnology and pharmaceutical industry. Large and small private sector organizations therefore stand to benefit from our research. Our current user network includes the UK subsidiary of Takeda; a large Japanese pharmaceutical house and the head of the transgenic core facility at a charity funded research centre. These are representative beneficiaries of our research. The field of transgenesis is a huge one and the benefits of our research will be felt widely. The major immediate benefit of our research has been and will be the ability to generate animal models of human disease more readily and more precisely than is currently possible. The development of these techniques will enhance the competitiveness of the UK biotech and pharmaceutical sector. The involvement of Takeda proves that this is so and, in this particular instance, will help anchor Takeda into the UK research environment. The benefits to the biotech and pharmaceutical industry will be realised on a time scale of one-three years. The speed with which other fields which use genome modification take up our technology is harder to envisage but is likely to be over the three-ten year period. In addition, phiC31 and phiBT1 integrases are from phages that infect Streptomyces sp, major antibiotic producers and thus at the forefront of the fight against infectious diseases. We have generated widely used vectors that others use for manipulation of antibiotic biosynthesis pathways and the generation of novel antibiotics. We plan to work up two new Streptomyces integrases that can, with little extra work, be made into novel vectors. Our staff will acquire experience in genome modification that will equip them to work in corresponding areas of the biotechnology and pharmaceutical industry. In the past there has been extensive movement of staff between academic labs and the private sector users of genome modification tools. This will continue. Our impact strategy will build upon pre-existing interactions with groups that are already using reagents that we have established during earlier BBSRC funded research. These groups are based in the commercial public sector, in an academic lab and in a core service laboratory of a charity funded research centre. The aim of these interactions has been to move tools and techniques developed by us into the laboratories of the respective users. We will not focus exclusively on the commercial public sector in our impact activities because in the genome manipulation field there is a lot of movement backwards and forwards between academic, service and commercial groups. In order to maximize impact we will organize a series of three one day workshops spread over the three years of the grant. The PIs and post-docs will present results of our work so far (ie pre-publication). We will encourage other attendees of the workshop to do likewise and to outline as far as possible the goals of their genome engineering activities and the technical problems they are encountering. We envisage that these interactions will help formalize the pre-existing informal network of those interested in genome engineering. We will establish a UK genome engineering club to encourage dissemination of genome manipulation and modification tools. This genome engineering club will expand the user group If we invent new patentable techniques during the course of our work then we will secure appropriate protection using the IP departments of our respective universities.