近日，美国西雅图合成生物学中心教授Jay Shendure及其小组研究出用于哺乳动物基因组工程和分子记录的启动子和gRNA支架部件清单。该项研究成果发表在2025年11月11日出版的《自然—生物技术》上。

在这里，课题组设计了现存的、祖先的、诱变的或小型化的聚合酶III启动子和引导RNA （gRNA）支架变体的文库，并量化了它们通过多重引物编辑介导哺乳动物基因组精确编辑的能力。该课题组确定了在人类和非主题细胞系中可重复编辑的部分，包括数百个比通常主题序列更具活性的部分。饱和诱变筛选鉴定耐受性序列变异，进一步增强序列多样性。在分子记录的应用中，研究小组设计了一个“十键”阵列，在哺乳动物细胞中，根据组成部分的活性预测，实现了pegRNAs的平衡活性。本文报道的数据将有助于设计合成的基因座编码阵列，这些基因座编码阵列具有可预测的、不同水平的活性，可用于多路扰动、生物记录和复杂的遗传回路。

据了解，微生物基因工程序列的标准化“部件清单”对于合成生物学的进步是不可或缺的，但是哺乳动物系统的类似部件却很少。

附：英文原文

Title: A parts list of promoters and gRNA scaffolds for mammalian genome engineering and molecular recording

Author: McDiarmid, Troy A., Taylor, Megan L., Chen, Wei, Chardon, Florence M., Choi, Junhong, Liao, Hanna, Li, Xiaoyi, Kim, Haedong, Lalanne, Jean-Benot, Li, Tony, Nathans, Jenny F., Martin, Beth K., Knuth, Jordan, Coradini, Alessandro L. V., Gray, Jesse M., Pinglay, Sudarshan, Shendure, Jay

Issue&Volume: 2025-11-11

Abstract: A standardized ‘parts list’ of sequences for genetic engineering of microbes has been indispensable to progress in synthetic biology, but few analogous parts exist for mammalian systems. Here we design libraries of extant, ancestral, mutagenized or miniaturized variants of polymerase III promoters and guide RNA (gRNA) scaffolds and quantify their abilities to mediate precise edits to the mammalian genome through multiplex prime editing. We identify thousands of parts for reproducible editing in human and mouse cell lines, including hundreds with greater activity than commonly used sequences. Saturation mutagenesis screens identify tolerated sequence variants that further enhance sequence diversity. In an application to molecular recording, we design a ‘ten key’ array that, in mammalian cells, achieves balanced activity of pegRNAs as predicted by the activity of the component parts. The data reported here will aid the design of synthetic loci encoding arrays of gRNAs exhibiting predictable, differentiated levels of activity for applications in multiplexed perturbation, biological recorders and complex genetic circuits.

DOI: 10.1038/s41587-025-02896-2

Source: https://www.nature.com/articles/s41587-025-02896-2