Targeted mutagenesis in rice using CRISPR-Cas system

By Jin Miao, Dongshu Guo, Jinzhe Zhang, Qingpei Huang, Genji Qin, Xin Zhang, J. Wan, H. Gu, Li-jia Qu. Cell Research, 2013

803 citations recorded by the source corpus.

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 …

Subfields: Aging, Business and International Management, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/e8ad95c8f22c1db8d7a46bcd5def5e790d3873bb

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Molecular Biology

1. R. Fischer
2. Daniel F. Voytas
3. L. Bortesi
4. Caixia Gao
5. Yong‐Bi Fu

Citations in the corpus

• A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. (2015, background): “CRISPR/Cas9 editing systems have enabled genomic targeting in many organisms, including plants (Cong et al., 2013; Jiang et al., 2013a, 2013b; Li et al., 2013; Mao et al., 2013; Miao et al., 2013; Shan et al., 2013b; Xie and Yang, 2013; Fauser et al., 2014; Zhang et al., 2014; Zhou et al., 2014).”
• Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation (2015, neutral): “system provides an alternative method [17–44].”
• The Sequence of 1504 Mutants in the Model Rice Variety Kitaake Facilitates Rapid Functional Genomic Studies (2017, background): “, 2012), CRISPR/Cas9 genome editing (Jiang et al., 2013; Miao et al., 2013; Xie et al., 2015), chemical induction, such as ethyl methanesulfonate (EMS) (Henry et al.”
• A CRISPR/Cas9 toolkit for multiplex genome editing in plants (2014, methodology): “Agrobacterium-mediated transformation is a routine method used to generate transgenic plants, and a few binary vectors have been developed to deliver the CRISPR/Cas9 system into plant genomes via this method [15,20,23,24,33-40].”
• Enhanced Rice Blast Resistance by CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor Gene OsERF922 (2016, result): “0%, similar to those previously reported for CRISPR/Cas9-induced mutations in rice [31, 35, 38, 39].”
• The CRISPR/Cas9 system for plant genome editing and beyond. (2015, background): “The CRISPR/Cas9 technology is therefore more versatile for genome editing in plants generally but particularly suitable for monocots with high genomic GC content such as rice (Miao et al., 2013).”
• A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation1[OPEN] (2015, background): “After 128 initial reports, many studies quickly announced successful and effective CRISPR/Cas9 129 mediated genome editing in plants (Feng et al., 2013; Jiang et al., 2013; Li et al., 2013; Mao et 130 al., 2013; Miao et al., 2013; Nekrasov et al., 2013; Shan et al., 2013; Xie and Yang, 2013; 131 Fau”