National Key Laboratory of Crop Genetic Improvement and Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, People's Republic of China.
Current affiliation: Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, People's Republic of China.
Curr Protoc. 2023 Sep;3(9):e905. doi: 10.1002/cpz1.905.
CRISPR/Cas9 genome editing is a revolutionary technology for plant functional genomics and crop breeding. In this system, the Cas9 nuclease is directed by a guide RNA (gRNA) to cut the DNA target and introduce mutation through error-prone DNA break repair. Owing to its simplicity, CRISPR/Cas9-mediated targeted gene knockout is widely used for high-throughput genetic screening in animal cell cultures and bacteria. However, high-throughput genetic screening using CRISPR/Cas9 is still challenging in plants. We recently established a new approach, named the FLASH genome editing pipeline, to construct an arrayed CRISPR library in plants. In this pipeline, a set of 12 PCR fragments with different lengths (referred to as FLASH tags) are used to index the Cas9/gRNA vectors. Subsequently, a mixture of 12 Agrobacterium strains, in which each strain contained a FLASH-tag indexed vector, was transformed into rice plants. As a result, a unique link between the target gene/gRNA and FLASH tag is generated, which allows reading gRNA information in bacterial strains and gene-edited plants using regular PCR and gel electrophoresis. This protocol includes step-by-step instructions for gRNA design, high throughput assembly of FLASH-tag indexed Cas9/gRNA plasmids, Agrobacterium-mediated transformation of 12 indexed plasmids, and fast assignment of target gene information in primary transformants. The arrayed CRISPR library described here is suitable for small- to large-scale genetic screening and allows fast and comprehensive gene function discovery in plants. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Assembly of FLASH-tag-indexed Cas9/gRNA plasmids Basic Protocol 2: Preparation of the Cas9/gRNA plasmid library Basic Protocol 3: Library preparation of Agrobacterium strains and mixing FLASH-tag indexed strains Basic Protocol 4: Grouped transformation and assignments of gRNA information of gene-edited plants.
CRISPR/Cas9 基因组编辑是植物功能基因组学和作物育种的革命性技术。在该系统中，Cas9 核酸酶由向导 RNA（gRNA）引导，切割 DNA 靶标，并通过易错 DNA 断裂修复引入突变。由于其简单性，CRISPR/Cas9 介导的靶向基因敲除被广泛用于动物细胞培养物和细菌中的高通量遗传筛选。然而，在植物中使用 CRISPR/Cas9 进行高通量遗传筛选仍然具有挑战性。我们最近建立了一种新方法，称为 FLASH 基因组编辑管道，用于在植物中构建阵列 CRISPR 文库。在该管道中，使用一组 12 个不同长度的 PCR 片段（称为 FLASH 标签）来标记 Cas9/gRNA 载体。随后，将包含 12 个农杆菌菌株的混合物转化到水稻植株中，其中每个菌株都含有一个 FLASH 标签标记的载体。结果，在靶基因/gRNA 和 FLASH 标签之间产生了独特的联系，这使得可以使用常规 PCR 和凝胶电泳在细菌菌株和基因编辑植物中读取 gRNA 信息。该方案包括 gRNA 设计、高通量组装 FLASH 标记的 Cas9/gRNA 质粒、12 个标记质粒的农杆菌介导转化以及初级转化体中靶基因信息的快速分配的分步说明。这里描述的阵列 CRISPR 文库适用于小规模到大规模的遗传筛选，并允许在植物中快速全面地发现基因功能。©2023Wiley Periodicals LLC. 基本方案 1：组装 FLASH 标记的 Cas9/gRNA 质粒 基本方案 2：Cas9/gRNA 质粒文库的制备 基本方案 3：农杆菌菌株文库的制备和混合 FLASH 标记的菌株 基本方案 4：分组转化和编辑植物 gRNA 信息的分配。
