Application of AI-Designed OpenCRISPR-1 for Highly Efficient Gene Editing in Soybean and Nicotiana benthamiana

The widespread application of CRISPR/Cas genome editing for commercial crop improvement is currently hindered by a complex and restrictive intellectual property (IP) landscape. The recent development of OpenCRISPR-1, a fully AI-designed and open-source Cas9-like nuclease, provides a promising, IP-unencumbered alternative; however, its efficacy in dicotyledonous plants remains largely uncharacterized. Here, we report the successful adaptation of the OpenCRISPR-1 system for highly efficient targeted mutagenesis in dicots. We constructed a plant-optimized binary vector (pBSE-OpenCRISPR-1) and validated its editing capability across two species. In soybean (Glycine max), targeting the GmFAD2-1B gene via an Agrobacterium rhizogenes-mediated hairy root transformation system yielded a robust mutation rate of approximately 50%. In Nicotiana benthamiana, stable Agrobacterium-mediated transformation targeting the phytoene desaturase homologs (NbPDSa/b) achieved a 75% editing efficiency in T0 lines, with up to 13.8% of events displaying complete homozygous or biallelic mutations and the corresponding visible albino phenotypes. Deep amplicon and Sanger sequencing revealed a characteristic mutation profile dominated by 1-bp insertions and small deletions occurring two to three nucleotides upstream of the PAM. These results demonstrate that the AI-designed OpenCRISPR-1 system is a highly active and versatile nuclease for dicot genome engineering, offering a powerful, commercially unencumbered tool to accelerate global crop trait improvement.