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Enzymatic processing of DNA containing tandem dihydrouracil by endonucleases III and VIII

Nucleic Acids Research
Oxford University Press
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  • Agricultural Science
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OP-NARE130781 1..12 Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice Wenzhi Jiang1, Huanbin Zhou2, Honghao Bi2, Michael Fromm3, Bing Yang2 and Donald P. Weeks1,* 1Department of Biochemistry, University of Nebraska, Lincoln, NE 68588, USA, 2Deparment of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA and 3Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE 68588, USA Received July 13, 2013; Accepted August 12, 2013 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 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 mu- tagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight suscep- tibility 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. INTRODUCTION The ability to target specific genes for knockout or re- placemen

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