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.
Original language | English |
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Pages (from-to) | 207-221 |
Number of pages | 15 |
Journal | Plant Molecular Biology |
Volume | 82 |
Issue number | 3 |
DOIs | |
Publication status | Published - Jun 2013 |
Funding
EU-FP7 TRACTAR grant, European Research CouncilWe thank Avishai Mor from the laboratory of Robert Fluhr in the Department of Plant Sciences at the Weizmann Institute of Science for his help with imaging bioluminescent luciferase activity. We also thank all members of Avraham A. Levy's laboratory in the Department of Plant Sciences at the Weizmann Institute of Science, especially Assaf Gavish for his help infiltrating N. benthamiana plants with Agrobacterium, followed by assaying for LUC and REN activities. This work was funded by an EU-FP7 TRACTAR grant from the European Research Council.
All Science Journal Classification (ASJC) codes
- Genetics
- Agronomy and Crop Science
- Plant Science