Callus and endosperm green fluorescence reporter-assisted selection system in maize CRISPR/Cas9 gene editing

Genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has emerged as a promising approach for functional gene analysis and genetic improvement. Since stable transformation remains the primary method for implementing this system, the ultimate goal in crop breeding programs would require the selection of transgene-free plants with the CRISPR/Cas expression cassette removed.

In this study, we developed an endosperm-specific fluorescence reporter-assisted selection system for CRISPR/Cas9 gene editing (pAZS22-eGFP/CRISPR/Cas9) in maize (Zea mays L.), utilizing enhanced green fluorescent protein (eGFP) expressed specifically in the endosperm to facilitate the easy identification of transgenic and transgene-free plants from the T₁ generation on. In addition, the 22 kDa alpha zein (z1C1_10) promoter from maize, employed in this system, has been shown to be active in both callus and endosperm, thereby being able to enhance the accuracy of transformant identification during the tissue culture process by reducing false positives compared to the traditional selective media methods. Our studies targeting the ZmSnRK2.1 or Dwarf1 (D1) genes demonstrated a reasonable editing efficiency, with rates ranging from 56.3% for T₀ plants targeting ZmSnRK2.1, to 87.5% and 100% for T₁ plants targeting D1 and ZmSnRK2.1, respectively. In addition, we successfully identified 1 transgene-free homozygous d1 mutant in the T₁ generation and 7 transgene-free homozygous snrk2.1 mutants in the T₂ generation.

The pAZS22-eGFP/CRISPR/Cas9 system provides an efficient tool for gene editing, transformant selection and transgene status identification in maize breeding.