A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants
| Autor: | Liu Wei, Xingliang Ma, Rongxin Shen, Jingxin Guo, Qinlong Zhu, Zhi Wang, Letian Chen, Yan Chen, Shuifu Chen, Bin Wang, Xiucai Zhao, Yao-Guang Liu, Lin Yuru, Yongyao Xie, Zhongfang Yang, Rong Qiu, Yuanling Chen, Heying Li, Qunyu Zhang, Zhicheng Dong |
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| Rok vydání: | 2015 |
| Předmět: |
Genetic Vectors
Molecular Sequence Data Arabidopsis Inheritance Patterns Plant Science Gene mutation Biology medicine.disease_cause Genes Plant Genome Genome editing Gene Expression Regulation Plant medicine Gene family CRISPR Gene Molecular Biology Alleles Genetics Mutation Base Sequence Cas9 food and beverages Oryza Plants Genetically Modified Phenotype RNA Editing CRISPR-Cas Systems Genome Plant RNA Guide Kinetoplastida |
| Zdroj: | Molecular Plant. 8(8):1274-1284 |
| ISSN: | 1674-2052 |
| DOI: | 10.1016/j.molp.2015.04.007 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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