title: A rapid assay to quantify the cleavage efficiency of custom-designed nucleases in planta.
abstract: Custom-designed nucleases are a promising technology for genome editing through the catalysis of double-strand DNA breaks within target loci and subsequent repair by the host cell, which can result in targeted mutagenesis or gene replacement. Implementing this new technology requires a rapid means to determine the cleavage efficiency of these custom-designed proteins in planta. Here we present such an assay that is based on cleavage-dependent luciferase gene correction as part of a transient dual-luciferase(®) reporter (Promega) expression system. This assay consists of co-infiltrating Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains: one contains the target sequence embedded within a luciferase reporter gene and the second strain contains the custom-designed nuclease gene(s). We compared repair following site-specific nuclease digestion through non-homologous DNA end-joining, as opposed to single strand DNA annealing, as a means to restore an out-of-frame luciferase gene cleavage-reporter construct. We show, using luminometer measurements and bioluminescence imaging, that the assay for non-homologous end-joining is sensitive, quantitative, reproducible and rapid in estimating custom-designed nucleases' cleavage efficiency. We detected cleavage by two out of three transcription activator-like effector nucleases that we custom-designed for targets in the Arabidopsis CRUCIFERIN3 gene, and we compared with the well-established 'QQR' zinc-finger nuclease. The assay we report requires only standard equipment and basic plant molecular biology techniques, and it can be carried out within a few days. Different types of custom-designed nucleases can be preliminarily tested in our assay system before their downstream application in plant genome editing.
authors.affiliation: Department of Plant Sciences, the Weizmann Institute of Science, P.O. Box 26, 76100 Rehovot, Israel. Ross.Johnson@Weizmann.ac.il
authors.forename: Ross A
authors.initials: RA
authors.lastname: Johnson
authors.forename: Vyacheslav
authors.initials: V
authors.lastname: Gurevich
authors.forename: Avraham A
authors.initials: AA
authors.lastname: Levy
mesh_terms: D001483:Base Sequence; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004260:DNA Repair; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D030821:Plants, Genetically Modified; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics / Q000378:metabolism
chemical_list: D018744:DNA, Plant; D010940:Plant Proteins; D008156:Luciferases; D004720:Endonucleases