Department of Chemical & Biomolecular Engineering, North Carolina State University, Raleigh, NC, 27695-7905, USA.
Helmholtz Institute for RNA-based Infection Research (HIRI)/Helmholtz Centre for Infection Research (HZI), 97080, Würzburg, Germany.
Nat Commun. 2021 Jan 22;12(1):555. doi: 10.1038/s41467-020-20633-y.
The ever-expanding set of CRISPR technologies and their programmable RNA-guided nucleases exhibit remarkable flexibility in DNA targeting. However, this flexibility comes with an ever-present constraint: the requirement for a protospacer adjacent motif (PAM) flanking each target. While PAMs play an essential role in self/nonself discrimination by CRISPR-Cas immune systems, this constraint has launched a far-reaching expedition for nucleases with relaxed PAM requirements. Here, we review ongoing efforts toward realizing PAM-free nucleases through natural ortholog mining and protein engineering. We also address potential consequences of fully eliminating PAM recognition and instead propose an alternative nuclease repertoire covering all possible PAM sequences.
不断扩展的 CRISPR 技术及其可编程 RNA 引导的核酸酶在 DNA 靶向方面表现出显著的灵活性。然而，这种灵活性伴随着一个始终存在的限制：每个目标侧翼都需要一个前导间隔相邻基序 (PAM)。虽然 PAMs 在 CRISPR-Cas 免疫系统的自我/非自我识别中发挥着重要作用，但这一限制促使人们广泛探索具有宽松 PAM 要求的核酸酶。在这里，我们通过天然同源物挖掘和蛋白质工程来回顾实现无 PAM 核酸酶的进展。我们还讨论了完全消除 PAM 识别的潜在后果，并提出了一个替代的核酸酶库，涵盖所有可能的 PAM 序列。
