小柯机器人

近日，美国莱斯大学Isaac B. Hilton研究小组发现，紧凑设计的人类机械敏感转录激活模块可实现强效、多用途的合成转录控制。2023年10月9日，国际知名学术期刊《自然—方法学》在线发表了这一成果。

研究人员表示，工程化转录激活结构域（TAD）与可编程DNA结合平台的结合彻底改变了合成转录控制。尽管基于CRISPR-Cas的可编程转录激活（CRISPRa）技术最近取得了进展，但这些系统中使用的TAD通常包含耐受性较差的元件，而且/或者对于许多应用来说过于庞大。

研究人员定义并优化了由人类机械敏感转录因子构建的最小TAD。研究人员利用这些元件构建了强效、紧凑的多组转录激活模块（MSN、NMS和eN3x9），并构建了CRISPR-dCas9募集增强激活模块（CRISPR-DREAM）平台。研究人员发现，CRISPR-DREAM在哺乳动物细胞类型中具有特异性和稳健性，能有效刺激不同调控位点的转录。研究人员还发现，MSN和NMS可跨I、II和V型CRISPR系统、转录激活剂样效应物和锌指蛋白。

此外，作为概念验证，研究人员使用dCas9-NMS将人类成纤维细胞高效重编程为诱导多能干细胞，并证明机械敏感转录因子TAD在具有重要治疗作用的人类原代细胞类型中具有良好的疗效和耐受性。最后，研究人员利用这些工程化TAD结构紧凑、功效强大的特点，构建了双重和多合一CRISPRa AAV系统。总之，这些小巧的人类TAD、融合模块和递送架构对于生物医学应用中的合成转录控制非常有价值。

附：英文原文

Title: Compact engineered human mechanosensitive transactivation modules enable potent and versatile synthetic transcriptional control

Author: Mahata, Barun, Cabrera, Alan, Brenner, Daniel A., Guerra-Resendez, Rosa Selenia, Li, Jing, Goell, Jacob, Wang, Kaiyuan, Guo, Yannie, Escobar, Mario, Parthasarathy, Abinand Krishna, Szadowski, Hailey, Bedford, Guy, Reed, Daniel R., Kim, Sunghwan, Hilton, Isaac B.

Issue&Volume: 2023-10-09

Abstract: Engineered transactivation domains (TADs) combined with programmable DNA binding platforms have revolutionized synthetic transcriptional control. Despite recent progress in programmable CRISPR–Cas-based transactivation (CRISPRa) technologies, the TADs used in these systems often contain poorly tolerated elements and/or are prohibitively large for many applications. Here, we defined and optimized minimal TADs built from human mechanosensitive transcription factors. We used these components to construct potent and compact multipartite transactivation modules (MSN, NMS and eN3x9) and to build the CRISPR–dCas9 recruited enhanced activation module (CRISPR-DREAM) platform. We found that CRISPR-DREAM was specific and robust across mammalian cell types, and efficiently stimulated transcription from diverse regulatory loci. We also showed that MSN and NMS were portable across Type I, II and V CRISPR systems, transcription activator-like effectors and zinc finger proteins. Further, as proofs of concept, we used dCas9-NMS to efficiently reprogram human fibroblasts into induced pluripotent stem cells and demonstrated that mechanosensitive transcription factor TADs are efficacious and well tolerated in therapeutically important primary human cell types. Finally, we leveraged the compact and potent features of these engineered TADs to build dual and all-in-one CRISPRa AAV systems. Altogether, these compact human TADs, fusion modules and delivery architectures should be valuable for synthetic transcriptional control in biomedical applications.

DOI: 10.1038/s41592-023-02036-1

Source: https://www.nature.com/articles/s41592-023-02036-1

Nature Methods：《自然—方法学》，创刊于2004年。隶属于施普林格·自然出版集团，最新IF：47.99
官方网址：https://www.nature.com/nmeth/
投稿链接：https://mts-nmeth.nature.com/cgi-bin/main.plex