The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs from hydrobacteria to reconstruct a common ancestor, ReChb, characterized by near-PAMless targeting and the recognition of diverse nucleic acid activators and collateral substrates. ReChb shares 53% sequence identity with the closest Cas12a ortholog but no longer requires a T-rich PAM and can achieve genome editing in human cells at sites inaccessible to the natural FnCas12a or the engineered and PAM-flexible enAsCas12a. Furthermore, ReChb can be triggered not only by double-stranded DNA but also by single-stranded RNA and DNA targets, leading to non-specific collateral cleavage of all three nucleic acid substrates with similar efficiencies. Finally, tertiary and quaternary structures of ReChb obtained by cryogenic electron microscopy reveal the molecular details underlying its expanded biophysical activities. Overall, ReChb expands the application space of Cas12a nucleases and underscores the potential of ASR for enhancing CRISPR technologies.

Cas12a 核酸酶的特性限制了可接近靶标的范围及其应用。在这项研究中，我们将祖先序列重建 （ASR） 应用于一组来自水细菌的 Cas12a 直系同源物，以重建一个共同的祖先 ReChb，其特征是近乎无 PAM的靶向和对不同核酸激活剂和侧支底物的识别。ReChb 与最接近的 Cas12a 直系同源物具有 53% 的序列同一性，但不再需要富含 T 的 PAM，并且可以在天然 FnCas12a 或工程化且 PAM 灵活的 enAsCas12a 无法接近的位点实现人类细胞的基因组编辑。此外，ReChb 不仅可以由双链 DNA 触发，还可以由单链 RNA 和 DNA 靶标触发，导致所有三种核酸底物的非特异性侧支切割，效率相似。最后，通过低温电子显微镜获得的 ReChb 的三级和四级结构揭示了其扩展生物物理活性背后的分子细节。总体而言，ReChb 扩展了 Cas12a 核酸酶的应用空间，并强调了 ASR 增强 CRISPR 技术的潜力。