| 1 | A FLASH pipeline for arrayed CRISPR library construction and the gene function discovery of rice receptor-like kinases显示文摘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 and demonstrate it is suitable for small-to large-scale genome editing in plants.This pipeline introduces artificial PCR fragment-length markers for distinguishing guide RNAs(gRNAs)(FLASH),and a group of 12 constructs harboring different FLASH tags are co-transformed into plants each time.The identities of gRNAs in Agrobacterium mixtures and trans-genic plants can therefore be read out by detecting the FLASH tags,a process that requires only conven-tional PCR and gel electrophoresis rather than sequencing.We generated an arrayed CRISPR library target-ing all 1,072 members of the receptor-like kinase(RLK)family in rice.One-shot transformation generated a mutant population that covers gRNAs targeting 955 RLKs,and 74.3%(710/955)of the target genes had three or more independent T0 lines.Our results indicate that the FLASH tags act as bona fide surrogates for the gRNAs and are tightly(92.1%)associated with frameshift mutations in the target genes.In addition,the FLASH pipeline allows for rapid identification of unintended editing events without corresponding T-DNA integrations and generates high-order mutants of closely related RLK genes.Furthermore,we showed that the RLK mutant library enables rapid discovery of defense-related RLK genes.This study introduces an effective pipeline for arrayed CRISPR library construction and provides genome-wide rice RLK mutant resources for functional genomics. | Kaiyuan Chen Runnan Ke Manman Du Yuqing Yi Yache Chen Xiaochun Wang Lu Yao Hao Liu Xin Hou Lizhong Xiong Yinong Yang Kabin Xie | 2022 | Molecular Plant2022，15，2： | 0 |
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