SCIENCE@home

Targeted mutagenesis in rice using CRISPR-Cas system

Jin Miao,Dongshu Guo,Jinzhe Zhang,Qingpei Huang,Genji Qin,Xin Zhang,J. Wan,H. Gu,Li-jia Qu

Published 2013 in Cell Research

ABSTRACT

Genome editing of model organisms is essential for gene function analysis and is thus critical for human health and agricultural production. The current technologies used for genome editing include ZFN (zinc-finger nuclease), meganucleases, TALEN (Transcription activa-tor-like effector nucleases), etc. [1]. These technologies can generate double stranded breaks (DSBs) to either disrupt gene function through generation of premature stop codons by non-homologous end joining (NHEJ) pathway, or to facilitate gene targeting through homolo-gous recombination (HR) with an incoming template. Recently, a new technology for genome editing, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) systems, has been developed [2]. CRISPR/Cas systems are adaptive defense systems in prokaryotic organisms to fight against alien nucleic acids [3]. The spacer sequences acquired from foreign DNA are positioned between host repeats, and transcribed together as CRISPR RNA (crRNA). In the type II CRISPR system, a single nuclease Cas9, guided by a dual-crRNA:tracrRNA, is sufficient to cleave cog-nate DNA homologous to the spacer [2]. Efficient cleav-age also requires the presence of protospacer adjacent motif (PAM) 5′-NGG-3′ following the spacer sequence. The dual-crRNA:tracrRNA has been further streamlined to a single RNA chimera, called sgRNA (single guide RNA) [2]. Compared with protein-guided technologies, CRISPR/Cas system is much easier to implement, as only short guide RNAs need to be customized to target the genes of interest. Up to now, the CRISPR/Cas system has been successfully applied to efficient genome editing in many eukaryotic organisms including human [1], mice [4], zebra fish [5], fly [6], worm [7], and yeast [8]. However, the application of CRISPR/Cas system in plants has not been reported. Rice (Oryza sativa L.) is a major staple crop in the grass family (Poaceae), feeding half of the world's population. Rice is also used as a model monocot plant for biological studies because it has a relatively small genome compared to other cereal crops and is easy to be manipulated genetically. We demonstrate in this study that the CRISPR/Cas technology can achieve efficient targeted mutagenesis in transgenic rice. Our work paves the way for large-scale genome editing in rice, which is important for quality improvement and yield increase of rice. To accommodate the CRISPR/Cas system to Agro-bacterium-mediated plant transformation, we designed Gateway TM binary T-DNA vectors for co-expression of CAS9 and guide RNA (either sgRNA or dual-crRNA:tracrRNA, see Figure 1A). Gene-specific spacer sequence was cloned into entry vectors for expression of guide RNA (Supplementary information, Figure S1 and …

PUBLICATION RECORD

CITATION MAP

EXTRACTION MAP

CLAIMS

  • No claims are published for this paper.

CONCEPTS

  • No concepts are published for this paper.

REFERENCES

Showing 1-15 of 15 references · Page 1 of 1

CITED BY

Showing 1-100 of 848 citing papers · Page 1 of 9

12349
Next