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

Literature Information ​

PMID25917172
JournalMolecular plant
Impact Factor24.1
JCR QuartileQ1
Publication Year2015
Times Cited954
KeywordsArabidopsis, CRISPR/Cas9, genome editing, rice, sequence-specific nucleases
Literature TypeJournal Article, Research Support, Non-U.S. Gov't
ISSN1674-2052
Pages1274-84
Issue8(8)
AuthorsXingliang Ma, Qunyu Zhang, Qinlong Zhu, Wei Liu, Yan Chen, Rong Qiu, Bin Wang, Zhongfang Yang, Heying Li, Yuru Lin, Yongyao Xie, Rongxin Shen, Shuifu Chen, Zhi Wang, Yuanling Chen, Jingxin Guo, Letian Chen, Xiucai Zhao, Zhicheng Dong, Yao-Guang Liu

TL;DR ​

This study presents a novel CRISPR/Cas9 vector system optimized for efficient multiplex genome editing in both monocot and dicot plants, achieving high mutation rates in rice and Arabidopsis. The approach enables the simultaneous targeting of multiple gene family members, enhancing the potential for functional studies and genetic improvements in plant research.

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

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.

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Primary Questions Addressed ​

  1. What are the potential applications of the robust CRISPR/Cas9 system in agricultural biotechnology?
  2. How does the efficiency of multiplex genome editing compare between monocot and dicot plants using this system?
  3. What challenges might researchers face when implementing this CRISPR/Cas9 system in different plant species?
  4. In what ways can this system be optimized further for higher efficiency or broader applications?
  5. How do the genetic outcomes of this multiplex editing approach influence plant breeding programs?

Key Findings ​

1. Research Background and Objective: The CRISPR/Cas9 genome editing technology has revolutionized genetic manipulation across various species, including plants. However, existing CRISPR/Cas9 systems for plant applications have predominantly focused on modifying one or a limited number of target sites, thereby constraining the potential for multiplex editing. The objective of this research was to develop a robust CRISPR/Cas9 vector system that enables convenient and high-efficiency multiplex genome editing in both monocot and dicot plants, thereby enhancing the capabilities for functional genomics and genetic improvement.

2. Main Methods and Findings: The researchers engineered a plant codon-optimized Cas9 gene and devised PCR-based protocols to generate multiple single guide RNA (sgRNA) expression cassettes rapidly. These cassettes could be assembled into binary CRISPR/Cas9 vectors using either Golden Gate ligation or Gibson Assembly in a single cloning step. The system was applied to rice, resulting in editing of 46 target sites with an impressive average mutation rate of 85.4%, predominantly achieving biallelic and homozygous mutations. It was determined that approximately 16% of these homozygous mutations arose via non-homologous end-joining (NHEJ) followed by homologous recombination. Additionally, in Arabidopsis T1 plants, uniform mutations were observed, including biallelic, heterozygous, homozygous, and chimeric forms. Notably, the resulting targeted mutations in both rice and Arabidopsis were confirmed to be heritable.

3. Core Conclusions: The developed CRISPR/Cas9 system represents a significant advancement in plant genome editing, allowing for the simultaneous targeting of multiple genes, gene family members, or sites within a single gene. The high efficiency and convenience of this system offer extensive possibilities for generating loss-of-function mutations and studying gene functions in a multiplex manner. The ability to achieve high mutation rates and heritable changes enhances the potential for functional genomics research and the development of genetically improved plant varieties.

4. Research Significance and Impact: This research has substantial implications for both basic plant science and agricultural biotechnology. By facilitating multiplex genome editing, the developed system can accelerate the functional characterization of gene families and metabolic pathways, providing insights into plant biology. Furthermore, the capability to create uniform genetic modifications improves the prospects for breeding programs aimed at crop improvement, enabling the development of varieties with enhanced traits such as disease resistance, yield, and abiotic stress tolerance. Overall, this work contributes to the growing toolkit for plant genetic engineering, opening new avenues for research and practical applications in agriculture.

Literatures Citing This Work ​

  1. Efficient CRISPR/Cas9-mediated Targeted Mutagenesis in Populus in the First Generation. - Di Fan;Tingting Liu;Chaofeng Li;Bo Jiao;Shuang Li;Yishu Hou;Keming Luo - Scientific reports (2015)
  2. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. - Zhi-Ping Wang;Hui-Li Xing;Li Dong;Hai-Yan Zhang;Chun-Yan Han;Xue-Chen Wang;Qi-Jun Chen - Genome biology (2015)
  3. Use of designer nucleases for targeted gene and genome editing in plants. - Donald P Weeks;Martin H Spalding;Bing Yang - Plant biotechnology journal (2016)
  4. A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation. - Levi G Lowder;Dengwei Zhang;Nicholas J Baltes;Joseph W Paul;Xu Tang;Xuelian Zheng;Daniel F Voytas;Tzung-Fu Hsieh;Yong Zhang;Yiping Qi - Plant physiology (2015)
  5. CLE Peptide Signaling and Crosstalk with Phytohormones and Environmental Stimuli. - Guodong Wang;Guohua Zhang;Mengyao Wu - Frontiers in plant science (2015)
  6. Revolutionizing plant biology: multiple ways of genome engineering by CRISPR/Cas. - Simon Schiml;Holger Puchta - Plant methods (2016)
  7. Exploiting the CRISPR/Cas9 System for Targeted Genome Mutagenesis in Petunia. - Bin Zhang;Xia Yang;Chunping Yang;Mingyang Li;Yulong Guo - Scientific reports (2016)
  8. A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. - Marta Vazquez-Vilar;Joan Miquel Bernabé-Orts;Asun Fernandez-Del-Carmen;Pello Ziarsolo;Jose Blanca;Antonio Granell;Diego Orzaez - Plant methods (2016)
  9. Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing. - Gang Liang;Huimin Zhang;Dengji Lou;Diqiu Yu - Scientific reports (2016)
  10. Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism. - Xuelian Zheng;Shixin Yang;Dengwei Zhang;Zhaohui Zhong;Xu Tang;Kejun Deng;Jianping Zhou;Yiping Qi;Yong Zhang - Plant cell reports (2016)

... (944 more literatures)

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