A yeast-based recombination assay for homing endonuclease activity

Abstract

Homing endonucleases (HEs) are natural enzymes that cleave long DNA target with a high specificity and trigger homologous recombination at the exact site of the break. Such mechanisms can thus be used for all the applications covered today by the generic name of "genome engineering": targeted sequence insertion, removal, or editing. However, before being able to address those applications, the engineering of HEs must be mastered so that any potential target would be efficiently and specifically recognized and cleaved. Working on the I-CreI model, we have developed a very powerful platform to generate HEs with new tailored specificity. We have put in place the first in vivo, functional, high throughput assay to generate I-CreI variants and measure their activity. We use semi-rational design combined with proprietary in silico predictions to design and synthesize I-CreI mutants that are tested for their capacity to induce homologous recombination in a yeast cell. The process has been standardized and robotized so that we can generate thousands of I-CreI derivatives, characterize their cleavage profile, and deliver them for further applications in the research, therapeutic, or agrobusiness fields. © Springer Science+Business Media New York 2014.