Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice
Abstract
The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-functional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5' coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.
- Authors:
-
- Univ. of Nebraska, Lincoln, NE (United States)
- Iowa State Univ., Ames, IA (United States)
- Publication Date:
- Research Org.:
- Univ. of Nebraska, Lincoln, NE (United States); Univ. of California, San Diego, CA (United States)
- Sponsoring Org.:
- USDOE Office of Energy Efficiency and Renewable Energy (EERE); National Science Foundation (NSF)
- OSTI Identifier:
- 1904805
- Grant/Contract Number:
- EE0001052; EE0003373; MCB-0952533; EPSCoR-1004094
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nucleic Acids Research
- Additional Journal Information:
- Journal Volume: 41; Journal Issue: 20; Journal ID: ISSN 0305-1048
- Publisher:
- Oxford University Press
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; synthetic biology; assembly cloning
Citation Formats
Jiang, Wenzhi, Zhou, Huanbin, Bi, Honghao, Fromm, Michael, Yang, Bing, and Weeks, Donald P. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. United States: N. p., 2013.
Web. doi:10.1093/nar/gkt780.
Jiang, Wenzhi, Zhou, Huanbin, Bi, Honghao, Fromm, Michael, Yang, Bing, & Weeks, Donald P. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. United States. https://doi.org/10.1093/nar/gkt780
Jiang, Wenzhi, Zhou, Huanbin, Bi, Honghao, Fromm, Michael, Yang, Bing, and Weeks, Donald P. Sat .
"Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice". United States. https://doi.org/10.1093/nar/gkt780. https://www.osti.gov/servlets/purl/1904805.
@article{osti_1904805,
title = {Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice},
author = {Jiang, Wenzhi and Zhou, Huanbin and Bi, Honghao and Fromm, Michael and Yang, Bing and Weeks, Donald P.},
abstractNote = {The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-functional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5' coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.},
doi = {10.1093/nar/gkt780},
journal = {Nucleic Acids Research},
number = 20,
volume = 41,
place = {United States},
year = {Sat Aug 31 00:00:00 EDT 2013},
month = {Sat Aug 31 00:00:00 EDT 2013}
}
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