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

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.

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation

“…While we were preparing our article, Xing et al (2014) and Ma et al (2015) reported a CRISPR/Cas9 toolkit for targeted mutagenesis in plants. They showed simultaneous knockout of multiple genes in Arabidopsis and rice, which, again, demonstrates the versatility and power of a multiplex CRISPR/Cas9 genome-editing system.…”

“…First, our comprehensive toolbox is designed for both genome editing and transcriptional regulation. To achieve this, we included four well-characterized Cas9 genes, including the one described by Ma et al (2015), all of which show high activity in plants (Feng et al, 2013;Li et al, 2013;Fauser et al, 2014). Aside from codon optimization, these Cas9 homologs differ from each in epitope tag fusions (Supplemental Fig.…”

“…S1), including three Cas9 genes that have been previously used in higher plants. They are plant codon-optimized Cas9 (pcoCas9, pYPQ150; Li et al, 2013), Arabidopsis codonoptimized Cas9 (AteCas9, pYPQ154; Fauser et al, 2014;Schiml et al, 2014), human codon-optimized Cas9 (hSpCas9, pYPQ158; Feng et al, 2013Feng et al, , 2014Mao et al, 2013;Zhang et al, 2014), and a plant codonoptimized Cas9 harboring enriched guanine-cytosine content within key 59 coding regions (Cas9p, pYPQ167; Ma et al, 2015). Also included within this module is the D10A nickase version of three out of these four Cas9 genes (pYPQ151, pYPQ155, and pYPQ159), although testing these nickases is not our focus here.…”

Selective gene dosage by CRISPR‐Cas9 genome editing in hexaploid Camelina sativa

“…The CRISPR‐Cas9 system can be used to achieve multiple mutagenesis by the expression of several sgRNAs targetting different genes (Lowder et al ., 2015; Ma et al ., 2015). The versatility and efficiency of CRISPR‐Cas9 system were also improved by Golden Braid and Gateway‐based vectors (Ma et al ., 2015; Vazquez‐Vilar et al ., 2016) and germ‐line‐specific Cas9 expression (Mao et al ., 2016). One major challenge is the ability of CRISPR‐Cas9 to induce mutations in polyploid genomes, in particular those with closely related subgenomes.…”

Generation of targeted mutant rice using a CRISPR‐Cpf1 system

“…Furthermore, the development and customization of plant CRISPR/Cas9 toolboxes or platforms result in more powerful molecular tools for fundamental research and crop breeding (Lowder et al ., 2015; Ma et al ., 2015; Mao et al ., 2016; Tang et al ., 2016; Wang et al ., 2015; Xie et al ., 2015; Xing et al ., 2014). Rice is the most frequently used crop species for testing and applying CRISPR‐Cas9 tools.…”