FLASH Genome Editing Pipeline: An Efficient and High‐Throughput Method to Construct Arrayed CRISPR Library for Plant Functional Genomics

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.