A simple test for the cleavage activity of customized endonucleases in plants

Nagaveni Budhagatapalli, Sindy Schedel, Maia Gurushidze, Stefanie Pencs, Stefan Hiekel, Twan Rutten, Stefan Kusch, Robert Morbitzer, Thomas Lahaye, Ralph Panstruga, Jochen Kumlehn, Goetz Hensel

Although customized endonucleases [transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs)] are known to be effective agents of 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 leaf epidermis transient expression test has been developed, based on reconstituting the translational reading frame of a mutated, non-functional yfp reporter gene. 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. Challenging either stable mutant alleles of a compromised version of gfp in tobacco and barley or the barley MLO gene with TALENs/RGENs confirmed the capacity to induce site-directed mutations. A convenient procedure to assay the cleavage activity of customized endonucleases has been established. The system is independent of the endonuclease platform and operates in both di- and monocotyledonous hosts. It not only enables the validation of a TALEN/RGEN's functionality prior to the creation of stable mutants, but also serves as a suitable tool to optimize the design of endonuclease constructs.