Serine integrases as a toolbox for genome editing

The development of a biological production platform for specialty chemicals requires on the one hand a profound knowledge of the organism’s genomics, its metabolism, and even its system biology, and on the other hand the availability of tools for small or large scale genetic adjustments. Fortunately, the creation of such a platform in Escherichia coli can lean on decades of fundamental and applied research (gene ontology, promoter-terminator databases, tool development for synthetic biology, …). Yet, the well-established methods for knocking out or knocking in genes or pathways of interest first described by Datsenko & Wanner in 2000 (using homologous recombination) are laborious and time consuming. These techniques typically only have ~10-6 efficiency, making selection markers inevitable. Moreover they suffer from a rapid decline in efficiency when large constructs are being inserted. Also CRISPR/Cas9, which is a hot topic in recent literature, is actually no more than a counter selection system which suffers from the same limitations as described above. Therefore we are developing a new genomic editing tool, genomic Serine Integrase Recombinational Assembly (gSIRA), exploiting serine integrases. The strength of gSIRA lies in the fact that it uses a completely different system for genomic integration and in addition these integrases are known for their ability to insert large constructs. First results are promising in which knock-ins have been made with 90% efficiency without the use of selection markers. In the near future the length-efficiency relation will be further investigated. In the end gSIRA will provide us with a new genomic editing tool which is able to insert entire pathways without the need of selection markers in a very efficient way