Jabalera Ylenia, Tascón Igor, Samperio Sara, López-Alonso Jorge P, Gonzalez-Lopez Monika, Aransay Ana M, Abascal-Palacios Guillermo, Beisel Chase L, Ubarretxena-Belandia Iban, Perez-Jimenez Raul
Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Derio, Spain.
Ikerbasque Foundation for Science, Bilbao, Spain.
Nat Biotechnol. 2024 Oct 31. doi: 10.1038/s41587-024-02461-3.
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其可作用靶点的范围及其应用。在本研究中，我们对一组来自水生细菌的Cas12a直系同源物应用祖先序列重建（ASR）来重建一个共同祖先ReChb，其特点是近乎无PAM靶向以及对多种核酸激活剂和附带底物的识别。ReChb与最接近的Cas12a直系同源物有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或经过工程改造且PAM灵活的enAsCas12a无法作用的位点在人类细胞中实现基因组编辑。此外，ReChb不仅可以被双链DNA触发，还可以被单链RNA和DNA靶点触发，从而以相似的效率对所有三种核酸底物进行非特异性附带切割。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的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，并突出了ASR在增强CRISPR技术方面的潜力。
