A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants
A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants
复制标题
DOI：
10.1016/j.molp.2015.04.007
复制
发表时间：
2015-08-03
期刊：
影响因子：
27.5
通讯作者：
Liu, Yao-Guang
中科院分区：
文献类型：
--
作者：
Ma, Xingliang;Zhang, Qunyu;Liu, Yao-Guang
CRISPR/Cas9基因组靶向系统已应用于多种物种。然而，大多数针对植物报道的CRISPR/Cas9系统只能修饰一个或几个靶位点。在这里，我们报告了一个强大的CRISPR/Cas9载体系统，利用植物密码子优化的Cas9基因，在单子叶和双子叶植物中进行方便和高效的多重基因组编辑。我们设计了基于PCR的程序来快速产生多个sgRNA表达盒，其可以通过Golden Gate连接或吉布森组装在一轮克隆中组装到二元CRISPR/Cas9载体中。利用该系统，我们在水稻中编辑了46个目标位点，平均突变率为85.4%，主要是双等位基因和纯合状态。我们推断，水稻中约16%的纯合突变是通过非同源末端连接机制，然后是基于同源重组的修复产生的。我们还获得了统一的双等位基因，杂合，纯合和嵌合突变拟南芥T-1植物。水稻和拟南芥中的靶向突变都是可遗传的。我们提供了T-0水稻和T-1拟南芥植物中功能丧失基因突变的例子，通过同时靶向一个基因家族的多个（多达8个）成员、生物合成途径中的多个基因或单个基因中的多个位点。该系统为研究植物多个基因和基因家族的功能提供了一个通用的工具箱，为基础研究和遗传改良提供了参考。
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 T-1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T-0 rice and T-1 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.