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Mol Plant. 2015 Aug;8(8):1274-84. doi: 10.1016/j.molp.2015.04.007. Epub 2015 Apr 24.

A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.

Author information

1
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou 510642, China; College of Life Sciences, South China Agricultural University, Guangzhou 510642, China.
2
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou 510642, China; Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, Guangzhou 510642, China; College of Life Sciences, South China Agricultural University, Guangzhou 510642, China.
3
Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, Guangzhou 510642, China; College of Life Sciences, South China Agricultural University, Guangzhou 510642, China.
4
Key Laboratory of South China Agriculture Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China.
5
College of Life Sciences, South China Agricultural University, Guangzhou 510642, China.
6
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou 510642, China; Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, Guangzhou 510642, China; Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou 510642, China; College of Life Sciences, South China Agricultural University, Guangzhou 510642, China.
7
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou 510642, China; Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, Guangzhou 510642, China; College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. Electronic address: ygliu@scau.edu.cn.

Abstract

CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.

KEYWORDS:

Arabidopsis; CRISPR/Cas9; genome editing; rice; sequence-specific nucleases

PMID:
25917172
DOI:
10.1016/j.molp.2015.04.007
[Indexed for MEDLINE]
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