Efficient CRISPR/Cas9-Mediated Gene Editing in Arabidopsis thaliana and Inheritance of Modified Genes in the T2 and T3 Generations

Jiang, WenZhi; Yang, Bing; Weeks, Donald P.

doi:10.1371/journal.pone.0099225

Efficient CRISPR/Cas9-Mediated Gene Editing in Arabidopsis thaliana and Inheritance of Modified Genes in the T2 and T3 Generations

Journal Article · Wed Jun 11 00:00:00 EDT 2014 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0099225· OSTI ID:1904806

Jiang, WenZhi ^[1]; Yang, Bing ^[2]; Weeks, Donald P. ^[3]

Univ. of Nebraska, Lincoln, NE (United States); Univ. of Nebraska, Lincoln, NE (United States)
Iowa State Univ., Ames, IA (United States)
Univ. of Nebraska, Lincoln, NE (United States)

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 GFP-negative 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.

View Accepted Manuscript (DOE)

Research Organization:: Univ. of California, San Diego, CA (United States); Univ. of Nebraska, Lincoln, NE (United States)

Sponsoring Organization:: National Science Foundation (NSF); USDOE Office of Energy Efficiency and Renewable Energy (EERE)

Grant/Contract Number:: EE0001052; EE0003373

OSTI ID:: 1904806

Journal Information:: PLoS ONE, Journal Name: PLoS ONE Journal Issue: 6 Vol. 9; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: English

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Efficient CRISPR/Cas9-Mediated Gene Editing in Arabidopsis thaliana and Inheritance of Modified Genes in the T2 and T3 Generations

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