A FLASH pipeline for arrayed CRISPR library construction and the gene function discovery of rice receptor-like kinases

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)−CRISPR-associated protein 9 (Cas9)-mediated gene editing is revolutionizing plant research and crop breeding. Here, we present an effective and streamlined pipeline for arrayed CRISPR library construction that is suitable for small- to large-scale genome editing in plants. This pipeline introduces artificial PCR f ragment- l ength m a rkers for guide RNAs (gRNAs) distingui sh ing (FLASH), and a group of 12 constructs harboring different FLASH tags are co-transformed into plants each time. Therefore, the identities of gRNAs in Agrobacterium mixtures and transgenic plants can be read out through detecting the FLASH tags which only requires conventional PCR and gel electrophoresis rather than sequencing. We generated an arrayed CRISPR library targeting all 1,072 members of receptor-like kinases (RLKs) family of rice. One-shot transformation generated a mutant population covering gRNAs targeting 955 RLKs, and 74.3% (710/955) of target genes had 3 or more independent T0 lines. Our results indicate that the FLASH tags bona fide surrogate the gRNAs and tightly (92.1%) associate with frameshift mutations of target genes. Additionally, the FLASH pipeline allows rapid identification of unintended editing events without corresponding T-DNA integrations and generates high-order mutants of closely related RLK genes. We also showed that the RLK mutant library enables fast discovery of defense-related RLK genes. Together, this study provides an effective pipeline for arrayed CRISPR library construction and reports genome-wide mutant resources of rice RLKs for functional genomics.