Comprehensive assessment of activity, specificity, and safety of hypercompact TnpB systems for gene editing

As the ancestor of CRISPR-Cas12 nucleases, TnpB represents the most compact gene editing tool currently available. Recent studies have identified multiple TnpB systems with gene editing activity in mammalian cells, and the potential of TnpB in treating diseases has been demonstrated in animal models. However, the editing characteristics of various TnpB systems, comparable to CRISPR tools, require more extensive investigation. Using a standardized evaluation framework, we conducted a thorough analysis of the editing properties of four TnpB variants alongside representative Cas12 and Cas9 tools. Overall, TnpBs exhibit intermediate editing activity and safety profiles among all tested systems, with ISYmu1 TnpB demonstrating a good performance in both editing activity and specificity. Considering its compact size, potent editing efficiency and high specificity, ISYmu1 TnpB represents a promising candidate for in vivo gene therapy applications. Overall design: We employed primer-extension-mediated sequencing (PEM-seq) to study the gene-editing outcomes for various TnpB systems and CRISPR-Cas nuclease, including: SpCas9, Nme2-C.NR, AsCas12a, CasMINI, ISDra2, ISDge10, ISAam1, ISYmu1. Twelve different editing sites were targeted for each nuclease.