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

Abstract 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