J Integr Plant Biol.

• Research Articles •    

Establishment of an efficient seed fluorescence reporter‐assisted CRISPR/Cas9 gene editing in maize

Yuanyuan Yan1,2, Jinjie Zhu1, Xiantao Qi1, Beijiu Cheng2, Changlin Liu1* and Chuanxiao Xie1*   

  1. 1Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing 100081, China
    2School of Life Sciences, Anhui Agricultural University, Hefei 230036, China

    *Correspondences: Changlin Liu (liuchanglin@caas.cn); Chuanxiao Xie (xiechuanxiao@caas.cn, Dr. Xie is fully responsible for distributions of all materials associated with this article)
  • Received:2021-01-29 Accepted:2021-02-27 Online:2021-03-02

Abstract: Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR‐associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While reporter‐assisted CRISPR/Cas systems can greatly facilitate the selection of genome‐edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)‐assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En‐SFR/Cas9), embryos (Em‐SFR/Cas9), or both tissues (Em/En‐SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐ SFR/Cas9 to identify plants not harboring the genome‐editing cassette but carrying the desired mutations at target genes in single genes or in small‐scale mutant libraries, and report on the successful generation of single‐target mutants and/or mutant libraries with En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐SFR/Cas9. SFR‐assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.

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