小柯机器人

美国哥伦比亚Samuel H. Sternberg研究团队利用CRISPR相关的转座酶在人类细胞中进行无双链断裂的DNA靶向整合。相关论文于2023年3月29日在线发表在《自然—生物技术》杂志上。

研究人员报告了一种在人类细胞中可编程整合大型DNA序列的方法，通过使用I-F型CRISPR相关转座酶（CAST）避免了双链断裂（DSB）的产生。研究人员通过蛋白质设计优化了QCascade复合物的DNA靶向，并利用AAA+ATP酶TnsC对QCascade靶向基因组位点的多价招募，开发了强效转录激活剂。在初步检测了基于质粒的整合后，研究人员又筛选了来自各种细菌宿主的15个CAST系统，并确定了一个来自假单胞菌的同源物，它表现出更好的活性并进一步提高了整合效率。

最后，研究人员发现，细菌的ClpX能以多个数量级增强基因组整合，可能是通过促进整合后CAST复合物的主动拆解，类似于它在Mu转座中的已知作用。这项工作强调了在人类细胞中重建复杂、多成分机制的能力，并为利用CRISPR相关转座酶进行真核生物基因组工程奠定了坚实基础。

据了解，传统的CRISPR-Cas9基因组工程会产生DSB，导致不良的副产物并降低产物的纯度。

附：英文原文

Title: Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases

Author: Lampe, George D., King, Rebeca T., Halpin-Healy, Tyler S., Klompe, Sanne E., Hogan, Marcus I., Vo, Phuc Leo H., Tang, Stephen, Chavez, Alejandro, Sternberg, Samuel H.

Issue&Volume: 2023-03-29

Abstract: Conventional genome engineering with CRISPR–Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an approach for programmable integration of large DNA sequences in human cells that avoids the generation of DSBs by using Type I-F CRISPR-associated transposases (CASTs). We optimized DNA targeting by the QCascade complex through protein design and developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase TnsC to genomic sites targeted by QCascade. After initial detection of plasmid-based integration, we screened 15 additional CAST systems from a wide range of bacterial hosts, identified a homolog from Pseudoalteromonas that exhibits improved activity and further increased integration efficiencies. Finally, we discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, likely by promoting active disassembly of the post-integration CAST complex, akin to its known role in Mu transposition. Our work highlights the ability to reconstitute complex, multi-component machineries in human cells and establishes a strong foundation to exploit CRISPR-associated transposases for eukaryotic genome engineering.

DOI: 10.1038/s41587-023-01748-1

Source: https://www.nature.com/articles/s41587-023-01748-1

Nature Biotechnology：《自然—生物技术》，创刊于1996年。隶属于施普林格·自然出版集团，最新IF：68.164
官方网址：https://www.nature.com/nbt/
投稿链接：https://mts-nbt.nature.com/cgi-bin/main.plex