Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia.
Nucleic Acids Res. 2023 Sep 22;51(17):9491-9506. doi: 10.1093/nar/gkad655.
Programmable site-specific nucleases promise to unlock myriad applications in basic biology research, biotechnology and gene therapy. Gene-editing systems have revolutionized our ability to engineer genomes across diverse eukaryotic species. However, key challenges, including delivery, specificity and targeting organellar genomes, pose barriers to translational applications. Here, we use peptide nucleic acids (PNAs) to facilitate precise DNA strand invasion and unwinding, enabling prokaryotic Argonaute (pAgo) proteins to specifically bind displaced single-stranded DNA and introduce site-specific double-strand breaks (DSBs) independent of the target sequence. We named this technology PNA-assisted pAgo editing (PNP editing) and determined key parameters for designing PNP editors to efficiently generate programable site-specific DSBs. Our design allows the simultaneous use of multiple PNP editors to generate multiple site-specific DSBs, thereby informing design considerations for potential in vitro and in vivo applications, including genome editing.
可编程的位点特异性核酸酶有望在基础生物学研究、生物技术和基因治疗中解锁无数的应用。基因编辑系统已经彻底改变了我们在不同真核生物物种中工程化基因组的能力。然而，包括递送、特异性和靶向细胞器基因组在内的关键挑战，对转化应用构成了障碍。在这里，我们使用肽核酸 (PNA) 来促进精确的 DNA 链入侵和展开，使原核 Argonaute (pAgo) 蛋白能够特异性地结合置换的单链 DNA，并在不依赖于靶序列的情况下引入位点特异性双链断裂 (DSB)。我们将这项技术命名为 PNA 辅助的 pAgo 编辑 (PNP 编辑)，并确定了设计 PNP 编辑以高效产生可编程的位点特异性 DSB 的关键参数。我们的设计允许同时使用多个 PNP 编辑器来产生多个位点特异性 DSB，从而为潜在的体外和体内应用（包括基因组编辑）提供设计考虑因素。
