Oh Youngbin, Lee Bora, Kim Hyeonjin, Kim Sang-Gyu
Department of Biological Sciences, KAIST, Daejeon, 34141 Republic of Korea.
Plant Methods. 2020 Mar 12;16:37. doi: 10.1186/s13007-020-00580-x. eCollection 2020.
The CRISPR system is composed of a Cas9 endonuclease (Cas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, Cas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.
We introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a Cas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco () protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by Cas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.
This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.
CRISPR系统由一个Cas9核酸内切酶（Cas9）和一个携带靶标特异性序列的单链引导RNA（gRNA）组成。理论上，Cas9蛋白能够切割与基因组中gRNA结合的多个靶位点。
我们引入了一种用于编辑植物基因组的无PCR多重gRNA克隆系统。该方法包括两个步骤：（1）将两个单链寡核苷酸片段的退火产物克隆到pGRNA载体中，这两个片段在每条链上均含有与靶标结合的互补序列，且位于tRNA和gRNA支架序列之间；（2）使用金门组装法将来自几个pGRNA载体的tRNA-gRNA单元与一个含有Cas9表达盒的植物双元载体进行组装。我们通过进行靶向深度测序验证了多重gRNA表达系统在野生烟草（）原生质体和转化植物中的编辑效率和模式。Cas9-gRNA的两个近端切割大大提高了编辑效率，并在两个切割位点之间诱导了大片段缺失。
这种多重gRNA表达系统能够高通量生产单个双元载体，并提高植物基因组编辑的效率。
