Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice

Wenzhi Jiang, Huanbin Zhou, Honghao Bi, Michael Fromm, Bing Yang, Donald P. Weeks

Research output: Contribution to journalArticle

517 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)e188
JournalNucleic acids research
Volume41
Issue number20
DOIs
StatePublished - Nov 1 2013

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
Sorghum
Arabidopsis
Tobacco
Genes
Fluorescence
DNA Sequence Analysis
CRISPR-Cas Systems
Arabidopsis Proteins
Agrobacterium tumefaciens
Gene Knockout Techniques
Genetic Engineering
Protoplasts
Streptococcus pyogenes
Zebrafish
Oryza
Genetic Promoter Regions
Mutagenesis
Diptera

ASJC Scopus subject areas

  • Genetics

Cite this

Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. / Jiang, Wenzhi; Zhou, Huanbin; Bi, Honghao; Fromm, Michael; Yang, Bing; Weeks, Donald P.

In: Nucleic acids research, Vol. 41, No. 20, 01.11.2013, p. e188.

Research output: Contribution to journalArticle

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. In: Nucleic acids research. 2013 ; Vol. 41, No. 20. pp. e188.
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