Clustered regularly interspaced short palindromic repeats (CRISPR)−CRISPR-associated protein 9 (Cas9)-mediated gene editing is revolutionizing plant research and crop breeding. To accelerate deciphering rice gene function, we developed FLASH pipeline for small-to-large scale CRISPR library construction. This pipeline introduces artificial PCR fragment-length markers for guide RNAs (gRNAs) distinguishing (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.
As an initial effort, 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.
Please click "Search" to check the status of plasmids and RLK mutant resources. A detailed instruction to request and use these seeds will be provided soon!
2021.9.26, first release a demo of RLK CRISPR library website.
2021.8, T1 seeds are available for RM1-RM12 groups.
Chen et al, A FLASH pipeline for arrayed CRISPR library construction and the gene function discovery of rice receptor-like kinases, Mol Plant, 2021, accepted.