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

Apr 29, 2015Molecular plant

A reliable CRISPR system for easy and efficient editing of multiple genes in grass and broadleaf plants

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Abstract

A CRISPR/Cas9 vector system achieved an average 85.4% mutation rate across 46 target sites in rice.

The developed system allows for high-efficiency multiplex genome editing in both monocot and dicot plants.
PCR-based procedures were utilized to quickly generate multiple sgRNA expression cassettes for vector assembly.
Homozygous mutations in rice were primarily produced through non-homologous end-joining followed by homologous recombination.
Uniform mutations, including biallelic, heterozygous, and chimeric types, were obtained in Arabidopsis T1 plants.
Targeted mutations in rice and Arabidopsis were shown to be heritable.
The system enables simultaneous targeting of multiple gene family members or genes within a biosynthetic pathway.

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

Abstract

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