4.7 Article

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

Journal

JOURNAL OF INTEGRATIVE PLANT BIOLOGY
Volume 63, Issue 9, Pages 1671-1680

Publisher

WILEY
DOI: 10.1111/jipb.13086

Keywords

CRISPR; Cas9; embryo‐ specific reporter; endosperm‐ specific reporter; genome editing; maize; seed fluorescence reporter

Funding

  1. National Science Foundation of China [31771808, 32001551]
  2. National Key R&D Program of China [2020YFE0202300]
  3. Key Area Research and Development Program of Guangdong Province [2018B020202008]
  4. Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences [S2021ZD03]

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The CRISPR/Cas9 genome editing technique has been established in maize using a seed fluorescence reporter system, allowing for the successful generation of desired mutants or mutant libraries. This approach may be particularly valuable for scenarios with low transformation frequency and has the potential to be expanded to other important monocot seed crops.
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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