TY - JOUR
T1 - Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice
AU - Jiang, Wenzhi
AU - Zhou, Huanbin
AU - Bi, Honghao
AU - Fromm, Michael
AU - Yang, Bing
AU - Weeks, Donald P.
N1 - Funding Information:
National Science Foundation (NSF) [MCB-0952533 and EPSCoR-1004094 to D.P.W.]; Department of Energy [DOE DE-EE0001052 and DOE CAB-COMM DOE DE-EE0003373 to D.P.W.]; Iowa State University Plant Science Institute Innovation Grant (to B.Y). Funding for open access charge: NSF.
PY - 2013/11
Y1 - 2013/11
N2 - 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 50 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 nearterm use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.
AB - 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 50 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 nearterm use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.
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U2 - 10.1093/nar/gkt780
DO - 10.1093/nar/gkt780
M3 - Article
C2 - 23999092
AN - SCOPUS:84886926151
SN - 0305-1048
VL - 41
SP - e188
JO - Nucleic acids research
JF - Nucleic acids research
IS - 20
ER -