AUTHOR=Rafi Mohammed , ElSiddig Mohamed , Aldarmaki Maitha , Al Nuaimi Mariam , George Suja , Amiri Khaled M. A. 

TITLE=Application of a multiplex CRISPR/Cas9 strategy for elimination of selection markers from transgenic plants

JOURNAL=Frontiers in Genome Editing

VOLUME=Volume 7 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/genome-editing/articles/10.3389/fgeed.2025.1633104

DOI=10.3389/fgeed.2025.1633104

ISSN=2673-3439

ABSTRACT=Selectable marker genes (SMGs) are essential for identifying transgenic plants but raise concerns regarding biosafety, regulatory compliance, and public acceptance. In this study, we used a CRISPR/Cas9-based strategy to eliminate the SMG from transgenic tobacco plants. Leaf discs from plants carrying DsRED (SMG) and aminoglycoside phosphotransferase (gene of interest, GOI) were re-transformed with a CRISPR vector containing four gRNAs designed to target both flanking regions of the SMG cassette. Approximately 20% of the regenerated shoots exhibited loss of red fluorescence, and PCR and sequencing analyses confirmed that about half of these carried a smaller amplicon, indicating a successful SMG excision efficiency of around 10%. Mutation analysis further revealed the presence of small indels at gRNA target sites, in addition to the deletion of SMG cassette. Quantitative real-time PCR (qPCR) analysis confirmed the absence of DsRED expression in SMG-deleted lines, while the Cas9 and GOI remained actively expressed. The SMG-free plants displayed normal growth, flowering, and seed production, indicating CRISPR marker excision had no adverse effects on plant development and fertility. In addition, Cas9-free, marker-free transgenic plants were recovered through segregation in T1 generation. This approach is adaptable to various transgenic plant species and provides a practical solution for generating marker-free transgenic crops, thereby enhancing their acceptance and commercialization.