The present study demonstrates the first CRISPR/Cas-mediated precise knock-in of the eGFP gene at the BABYBOOM2 (GN-BBM2) locus in banana cv. Grand Naine, facilitating the detection of editing events in early embryogenic developmental stages. Genome editing has accelerated crop improvement programs by introducing targeted and precise genetic modifications. Among different tools, CRISPR/Cas-based genome editing has been widely used for enabling mutations through double-stranded breaks (DSBs), repaired either by non-homologous end joining (NHEJ) for gene knockouts or homology-directed repair (HDR) to generate knock-in events. While gene knockouts are well established in banana, efficient knock-in remains a major challenge due to low HDR activity, sterility, and the vegetatively propagated nature of banana. In the present study, we report the first successful CRISPR/Cas-based gene knock-in editing in banana by targeting the BABYBOOM2 (BBM2) gene, which encodes a transcription factor involved in somatic embryogenesis. The enhanced green fluorescent protein (eGFP) gene was precisely inserted at the BBM2 locus in banana cv. Grand Naine to enable visual detection during embryogenesis. In vitro validation showed ~ 95% target cleavage efficiency of the selected gRNA. The PCR-based screening and shift-in amplicon size analyses confirmed three edited lines (#3, #11, and #14) harboring eGFP knock-in at the targeted locus. Sequencing of the amplicon from these lines further confirmed the precise knock-in events. Hence, this study establishes a foundation for precise knock-in-based genome modification in banana and opens new avenues for targeted trait improvement in this important clonally propagated crop.
