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

Published Date

3 August 2015, Vol.8(8):1274–1284, doi:10.1016/j.molp.2015.04.007

Research Article

Title 

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

• Author 

• Xingliang Ma ^1,4
• Qunyu Zhang ^1,2,4
• Qinlong Zhu ^2,4
• Wei Liu ^1,2,4
• Yan Chen ⁵
• Rong Qiu ^1,2,4
• Bin Wang ^1,2,4
• Zhongfang Yang ^1,2,4
• Heying Li ^1,2,4
• Yuru Lin ^1,2,4
• Yongyao Xie ^1,2,4
• Rongxin Shen^1,2,4
• Shuifu Chen ^1,2,4
• Zhi Wang ⁴
• Yuanling Chen ^1,2,4
• Jingxin Guo ^1,2,4
• Letian Chen ^1,2,3,4
• Xiucai Zhao ^1,2,4
• Zhicheng Dong ⁵
• Yao-Guang Liu ^1,2,4,,

• 1State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou 510642, China
• 2Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, Guangzhou 510642, China
• 3Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou 510642, China
• 4College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
• 5Key Laboratory of South China Agriculture Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China

Received 8 March 2015. Revised 10 April 2015. Accepted 15 April 2015. Available online 23 April 2015. Published: April 23, 2015

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.

Key words

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

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• PDF (2.7M)

Document S1. Supplemental Figures 1–7 and Supplemental Tables 1–8

• Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

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