Directed evolution of enzymes using in vitro compartmentalization (IVC) (2017)

Philipp Holliger and his team at the MRC Laboratory of Molecular Biology (LMB) have, over a number of years, developed an in vitro compartmentalization (IVC) technique using aqueous droplets of water-in-oil emulsions, which form cell-like compartments. These compartments are designed such that each is “clonal”, i.e. contains no more than a single cell or gene. Thus, both the genes (DNA) and their products (RNAs and/or proteins) are ‘trapped’ within the same compartment. By coupling the genotype (DNA) and phenotype (RNA, protein), in vitro compartmentalization allows the selection and evolution of phenotype.

The method can be used for high-throughput screening of proteins, peptides or functional RNAs with desired characteristics. Several modifications of the method allow for various types of selection under various conditions, for example, compartmentalised self-replication, allow for selection of enzymes with high activity or processivity, enzymes with relaxed or expanded substrate specificity, or low error rate. Enzymes can thus be selected for and tailored to specific applications.

Multiple patent applications have been filed protecting this technology, which have since been granted. Rights to the technology are offered on a non-exclusive basis and several licenses have been concluded. In 2017 MRC licensed the technology on a non-exclusive basis to a large biotech company who sells optimised enzymes for use in next-generation sequencing (NGS), as well as PCR and real-time PCR applications. Under the license agreement the company has the rights to use the technology to develop in vitro diagnostics.

The application of IVC in selection and evolution of polymerases exhibiting desired characteristics continues to be actively developed by the group at the LMB. The enzymes recently developed include a thermostable reverse transcriptase, which has been licensed on a non-exclusive basis to a company focused upon providing reagents for academic and industrial use. The portfolio of IP continues to attract commercial interest.