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

Kaiyuan Chen, Runnan Ke, Manman Du, Yuqing Yi, Yache Chen, Xiaochun Wang, Lu Yao, Hao Liu, Xin Hou, Lizhong Xiong, Yinong Yang, Kabin Xie

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

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 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 transgenic plants can therefore be read out by detecting the FLASH tags, a process that requires only conventional PCR and gel electrophoresis rather than sequencing. We generated an arrayed CRISPR library targeting 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.

Original languageEnglish (US)
Pages (from-to)243-257
Number of pages15
JournalMolecular plant
Volume15
Issue number2
DOIs
StatePublished - Feb 7 2022

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Plant Science

Fingerprint

Dive into the research topics of 'A FLASH pipeline for arrayed CRISPR library construction and the gene function discovery of rice receptor-like kinases'. Together they form a unique fingerprint.

Cite this