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Efficient CRISPR / Cas 9-Mediated Gene Editing in Arabidopsis thaliana and Inheritance of Modified Genes in the T 2 and T 3 Generations

The newly developed CRISPR/Cas9 system for targeted gene knockout or editing has recently been shown to function in plants in both transient expression systems as well as in primary T1 transgenic plants. However, stable transmission of genes modified by the Cas9/single guide RNA (sgRNA) system to the T2 generation and beyond has not been demonstrated. Here we provide extensive data demonstrating the efficiency of Cas9/sgRNA in causing modification of a chromosomally integrated target reporter gene during early development of transgenic Arabidopsis plants and inheritance of the modified gene in T2 and T3 progeny. Efficient conversion of a nonfunctional, out-of-frame GFP gene to a functional GFP gene was confirmed in T1 plants by the observation of green fluorescent signals in leaf tissues as well as the presence of mutagenized DNA sequences at the sgRNA target site within the GFP gene. All GFP-positive T1 transgenic plants and nearly all GFPnegative plants examined contained mutagenized GFP genes. Analyses of 42 individual T2 generation plants derived from 6 different T1 progenitor plants showed that 50% of T2 plants inherited a single T-DNA insert. The efficiency of the Cas9/ sgRNA system and stable inheritance of edited genes point to the promise of this system for facile editing of plant genes. Citation: Jiang W, Yang B, Weeks DP (2014) Efficient CRISPR/Cas9-Mediated Gene Editing in Arabidopsis thaliana and Inheritance of Modified Genes in the T2 and T3 Generations. PLoS ONE 9(6): e99225. doi:10.1371/journal.pone.0099225 Editor: Frederik Börnke, Leibniz-Institute for Vegetable and Ornamental Plants, Germany Received February 14, 2014; Accepted May 13, 2014; Published June 11, 2014 Copyright: 2014 Jiang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this research was provided, in part, by the National Science Foundation (MCB-0952533 and EPSCoR-1004094 to D.P.W.) and the Department of Energy (DOE DE-EE0001052 and DOE CAB-COMM DOE DE-EE0003373 to D.P.W.) and Iowa State University Plant Science Institute Innovation Grant (to B.Y.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dweeks1@unl.edu