Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice
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Open Access
- 31 August 2013
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 41 (20), e188
- https://doi.org/10.1093/nar/gkt780
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-fuctional, 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.Keywords
This publication has 47 references indexed in Scilit:
- One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome EngineeringCell, 2013
- Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryosCell Research, 2013
- Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene ExpressionCell, 2013
- Efficient TALEN-mediated gene knockout in livestockProceedings of the National Academy of Sciences, 2012
- RNA-guided genetic silencing systems in bacteria and archaeaNature, 2012
- High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleasesNature Methods, 2011
- Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotesNucleic Acids Research, 2011
- Efficient construction of sequence-specific TAL effectors for modulating mammalian transcriptionNature Biotechnology, 2011
- Targeted gene addition into a specified location in the human genome using designed zinc finger nucleasesProceedings of the National Academy of Sciences, 2007
- Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol componentsPlant Molecular Biology Reporter, 1997