Thornton, B. W.-L.; Weissman, R. F.; Tran, R. V.; Duong, B. T.; Rodriguez, J. E.; Terrace, C. I.; Groover, E. D.; Park, J.-U.; Tartaglia, J.; Doudna, J. A.; Savage, D.

TnpB is an evolutionarily diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Due to their small size and putative evolutionary relationship to Cas12s, TnpB holds significant potential for genome editing and mechanistic exploration. However, most TnpBs lack robust gene-editing activity, and unbiased profiling of mutational effects on editing activity has not been experimentally explored. Here, we mapped comprehensive sequence-function landscapes of a TnpB ribonucleoprotein and discovered many activating mutations in both the protein and RNA. Single-position changes in the RNA outperform existing variants, highlighting the utility of systematic RNA scaffold mutagenesis. Leveraging the mutational landscape of the TnpB protein, we identified enhanced protein variants from a combinatorial library of activating mutations, which increased editing levels in yeast and human cells relative to wild-type TnpB. In total, this study highlights unknown elements critical for regulation of endonuclease activity in both the TnpB protein and the RNA, and reveals a surprising amount of latent activity accessible through mutation.