CRISPR-associated transposases (CASTs) are an attractive candidate for genome editing applications, as they enable the insertion of large DNA cargoes without creating double-strand breaks. However, CAST systems have shown limited activity in human cells. In a paper published in Science, Witte et al. apply phage-assisted continuous evolution (PACE) to direct the rapid evolution of new CAST variants, acquiring a CAST system capable of efficiently integrating gene-size cargoes in human cells.

Iterative rounds of PACE yielded an evolved TnsB — a component of the Type I-F CAST transposition machinery — with integration efficiency in HEK cells more than 200-fold higher than that of the wild type. The evolved TnsB contained ten activity-enhancing mutations spanning multiple domains, suggesting that PACE optimized diverse functionalities to improve TnsB’s performance and that obtaining such a variant through rational protein engineering would have been unlikely. Notably, the evolved TnsB did not require supplementation with the accessory protein ClpX, a cytotoxic factor previously used to increase CAST editing efficiency.