西班牙巴斯克研究与技术联盟Raul Perez-Jimenez等研究人员合作团队发现，一个复活的Cas12a祖先扩展核酸编辑和检测的靶标访问和底物识别。相关论文于2024年10月31日在线发表于国际学术期刊《自然—生物技术》。

研究人员应用祖先序列重建（ASR）技术对一组来自水生细菌的Cas12a同源物以重建一个共同祖先ReChb，其特点是近无PAM靶向和对多样核酸激活物及伴随底物的识别。ReChb与最接近的Cas12a同源物的序列相似性为53%，但不再需要T丰富的PAM。并且能够在对天然FnCas12a或工程化、PAM灵活的enAsCas12a无法接触的位点上实现人类细胞的基因组编辑。此外，ReChb不仅可以被双链DNA激活，还可以被单链RNA和DNA靶标激活，导致对三种核酸底物的非特异性伴随切割，其效率相似。最后，通过冷冻电子显微镜获得的ReChb的三级和四级结构揭示了其扩展生物物理活动的分子细节。总体而言，ReChb扩展了Cas12a核酸酶的应用空间，并强调了ASR在增强CRISPR技术中的潜力。

据介绍，Cas12a核酸酶的特性限制了可接触靶标及其应用范围。

附：英文原文

Title: A resurrected ancestor of Cas12a expands target access and substrate recognition for nucleic acid editing and detection

Author: Jabalera, Ylenia, Tascn, Igor, Samperio, Sara, Lpez-Alonso, Jorge P., Gonzalez-Lopez, Monika, Aransay, Ana M., Abascal-Palacios, Guillermo, Beisel, Chase L., Ubarretxena-Belandia, Iban, Perez-Jimenez, Raul

Issue&Volume: 2024-10-31

Abstract: 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.

DOI: 10.1038/s41587-024-02461-3

Source: https://www.nature.com/articles/s41587-024-02461-3