Engineered TnpB genome editors for plants and human cells identified by ribonucleoprotein mutational scanning

Abstract TnpB is a diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Because of their small size and putative evolutionary relationship to CRISPR–Cas12, TnpB enzymes hold great potential for genome editing. However, most TnpBs lack robust gene-editing activity. Here, we mapped comprehensive sequence–function landscapes of a TnpB ribonucleoprotein using deep mutational scanning and we discovered activating mutations in both the RNA and the protein. Leveraging the protein’s mutational landscape, we constructed a combinatorial library of activating mutations, from which we identified two enhanced TnpB variants. These variants increased editing in human cells, Nicotania benthamiana , pepper and rice. While editing efficiencies varied by target site, engineered variants achieved up to 55% insertion and deletion frequencies (a 50-fold increase over wild type) in N . benthamiana , surpassing ISYmu1 (<7%), As Cas12f-HKRA (<9%) and other compact editors. These findings highlight elements critical for regulating TnpB endonuclease activity and demonstrate latent activity accessible through mutation.