NucleasesNucleases such as Cas9 and Cas12a have revolutionized genome editing by precisely targeting DNA for modification. These nucleases are considered to have evolved from ancestral proteins IscB and TnpB, respectively. Recent studies have demonstrated TnpB’s function as an RNA-guided nuclease in human cells. Notably, TnpB proteins are smaller than Cas9 and Cas12a, which facilitates their delivery into cells, especially via viral vectors, making TnpB a promising candidate for advanced genome editing tools in eukaryotes. We have recently optimized a system for editing plant genomes using the TnpB nuclease. Here, we present a step-by-step protocol for genome editing in rice and Arabidopsis using a rice codon-optimized TnpB derived from Deinococcus radiodurans (IsDra2TnpB). This TnpB-based method provides a highly efficient alternative to Cas9- and Cas12a-mediated editing, suitable for a wide range of plant species.