CRISP: A cost-efficient, high-throughput pipeline for sex-identification in Cannabis sativa

Hemp ( Cannabis sativa ) is a dioecious species whose glandular trichomes, found predominantly on female flowers, produce a variety of phytocannabinoids. Determining plant sex during the vegetative stage is not feasible, as only flowering plants allow accurate distinction. To efficiently utilize cultivation space and prevent unwanted pollination, it is therefore essential to reliably identify male hemp plants at an early developmental stage. Although numerous methods for sex identification in hemp have been reported, most rely on multi-step DNA extraction workflows and require costly equipment and consumables. Here, we present CRISP (Cannabis Rapid Identification of Sex by PCR), a simple, rapid, and cost-effective high-throughput approach. CRISP combines a simplified genomic DNA extraction via high-temperature chemical lysis with PCR detection of the male hemp marker SCAR323 in a 96-well format. We identified male seedlings from two cultivars with 100% accuracy, demonstrating the suitability of CRISP for large-scale sex identification in a highly cost-efficient manner.