A simple test system for customized endonucleases and homology-directed genome editing
G. HENSEL (1), N. Budhagatapalli (2), S. Schedel (2), S. Hiekel (2), T. Rutten (2), J. Kumlehn (2) (1) Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Plant Reproductive Biology Correns, Germany; (2) Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany

Although customized endonucleases (TALENs [transcription activator-like effector nucleases] and RGENs [RNA-guided endonucleases]) are known to be effective agents of site-directed mutagenesis in various host plants, newly designed endonuclease constructs require some pre-validation with respect to functionality before investing in the creation of stable transgenic plants. A simple, biolistics-based transient expression test has been developed, which indicates endonuclease cleavage activity via restitution of an out-of-frame yfp reporter gene in leaf epidermis. Quantification of mutation efficacy was made possible by co-bombarding the explant with a constitutive mCherry expression cassette, thereby allowing the ratio between the number of red and yellow fluorescing cells to serve as a metric for mutation efficiency. More sophisticated applications of programmable endonucleases involve the use of a DNA repair template facilitating homology-directed repair so as to create predefined rather than random DNA sequence modifications. Beside gene knock-out approaches, we have demonstrated that also a targeted allele conversion can be achieved in barley after TALEN- or RGEN-mediated double strand break induction by concomitant provision of a repair template leading to an intended exchange of a single amino acid which itself entails an altered protein function. This technology will be useful to functionally validate endonuclease constructs customized for genes involved in plant-microbe interactions.

Abstract Number: P9-258
Session Type: Poster