The Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 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 SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-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.

中文翻译：

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一种多重指导 RNA 表达系统及其在植物基因组工程中的功效

化脓性链球菌 CRISPR 系统由 Cas9 核酸内切酶 (SpCas9) 和含有靶标特异性序列的单链向导 RNA (gRNA) 组成。从理论上讲，SpCas9 蛋白可以切割与基因组中 gRNA 结合的尽可能多的靶向位点。我们介绍了一种用于编辑植物基因组的无 PCR 多 gRNA 克隆系统。该方法包括两个步骤： (1) 克隆 pGRNA 载体中 tRNA 和 gRNA 支架序列之间每条链上具有互补靶结合序列的两个单链寡核苷酸片段的退火产物；(2) 使用金门组装方法将来自多个 pGRNA 载体的 tRNA-gRNA 单元与包含 SpCas9 表达盒的植物二元载体组装在一起。我们通过进行靶向深度测序验证了野生烟草（Nicotiana attenuata）原生质体和转化植物中多重 gRNA 表达系统的编辑效率和模式。SpCas9-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的缺失。这种多重 gRNA 表达系统可实现单个二元载体的高通量生产，并提高植物基因组编辑的效率。