Multiplex Genome Editing Overcomes Photoperiod Sensitivity in Tropical Maize

Tropical maize is a rich source of genetic diversity that could enhance temperate maize breeding programs, but its sensitivity to long-day photoperiods, resulting in delayed flowering, limits its widespread use. To overcome this barrier, the Genome Engineering to Sustain Crop Improvement (GETSCI) project used CRISPR/Cas9 to mutate three flowering repressor genes, ZmCCT9, ZmCCT10 , and ZmRAP2 .7, in the tropical inbred Tzi8. A single sgRNA targeting the first exon of each target gene was combined with an excision cassette carrying the morphogenic genes Babyboom ( Bbm ) and Wuschel2 ( Wus2 ) to enable efficient transgenic plant regeneration. Transgenic plants carrying frameshift edits in each target gene were recovered, and subsequent crosses produced two non-transgenic genotypes: a double-edited zmcct10, zmrap2 . 7 line and a triple-edited zmcct9, zmcct10, zmrap2 . 7 line. Multiple flowering traits were measured for the edited genotypes and unedited Tzi8 inbred in short-day (Hawai‘i) and long-day (Iowa) field conditions. Both edited genotypes flowered significantly earlier than Tzi8 in both environments. Notably, under long-day conditions, flowering of the two edited lines overlapped with that of the temperate inbred B73, whereas Tzi8 did not. Together, these results demonstrate that targeted, multiplex gene editing can reduce photoperiod sensitivity in a tropical inbred, expanding access to previously untapped genetic diversity for temperate maize improvement.