植物基因组编辑的最大挑战是利用同源修复（HDR）的方式实现高效的多基因同时编辑。最近，植物基因编辑界的大牛Daniel F. Voytas构建了一个多功能的植物基因组编辑“工具箱”，可以对单双子叶植物基因进行高效的多重定点编辑和修饰。
该工具箱以TALEN和CRISPR/Cas9为基础，通过利用Golden Gate克隆载体快速的，模块化的实现单独或多重敲除以及大片段删除，从而达到基因的敲除、替换或转录修饰等多种基因组编辑的目的。此外，研究人员设计了网站工具可以使载体的选择和构建流程化(http://cfans-pmorrell.oit.umn.edu/CRISPR_Multiplex/)。该平台的一个优势是同时表达多个gRNA以提高多基因突变的效率。利用Csy4核糖核酸酶和tRNA加工酶在诱导突变的效果上是RNA聚合酶III的两倍，如果再用上TREX2核算外切酶，突变率将再增加2.5倍。试验证明，该系统通过表达12个gRNA实现了对6个基因的同时敲除。
该系统已在烟草、番茄、苜蓿、小麦和大麦中得到应用。这也是苜蓿基因编辑的首次成功报道。
Plant Cell. 2017 May 18.
A multi-purpose toolkit to enable advanced genome engineering in plants.
Author
Cermak T, Curtin SJ, Gil-Humanes J……Mathre JW, Greenstein RL, Voytas DF*.
*: Department of Genetics, CellBiology & Development and Center for Genome Engineering, University ofMinnesota, USA
Abstract
We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on TALENs and the CRISPR/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A web-based tool greatly stream lines vector selection and construction. One advantage of our platform is the use of the Csy4 ribonuclease and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing twelve gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III (Pol III) promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the TREX2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato, tobacco, Medicago, wheat and barley.
