小柯机器人

美国麻省理工学院Jonathan S. Gootenberg和Omar O. Abudayyeh共同合作，近期取得重要工作进展。他们研究发现，在不引入DNA双链断裂情况下，利用CRISPR定向整合酶能在基因组中插入大片段DNA序列。相关研究成果2022年11月24日在线发表于《自然—生物技术》杂志上。

研究人员开发了位点特异性靶向元件（PASTE）工具，该工具将逆转录酶和丝氨酸整合酶融合到CRISPR–Cas9内切酶上，用于靶向基因组招募和整合目的片段。研究人员在3种人类细胞、初级T细胞和未分裂初级人类肝细胞上的多个基因位点整合了约36kb的大片段DNA。为了增强PASTE功能，研究人员从宏基因组和工程直系同源物中发现了25614个丝氨酸整合酶和同源连接位点，它们具有更高的活性和更短的识别序列，以实现高效的可编程整合。

PASTE的编辑效率与基于同源重组和非同源末端连接修复的方法相似或超过这些方法，在非分裂细胞中也有活性，并且在体内脱靶率很低。PASTE扩大了基因组编辑的能力，允许在不依赖DNA修复途径的情况下进行复杂多样的大片段基因插入。

据介绍，在不引入DNA双链断裂的情况下，将大片段DNA整合到基因组中，仍然是基因编辑领域未解决的挑战。

附：英文原文

Title: Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases

Author: Yarnall, Matthew T. N., Ioannidi, Eleonora I., Schmitt-Ulms, Cian, Krajeski, Rohan N., Lim, Justin, Villiger, Lukas, Zhou, Wenyuan, Jiang, Kaiyi, Garushyants, Sofya K., Roberts, Nathaniel, Zhang, Liyang, Vakulskas, Christopher A., Walker, John A., Kadina, Anastasia P., Zepeda, Adrianna E., Holden, Kevin, Ma, Hong, Xie, Jun, Gao, Guangping, Foquet, Lander, Bial, Greg, Donnelly, Sara K., Miyata, Yoshinari, Radiloff, Daniel R., Henderson, Jordana M., Ujita, Andrew, Abudayyeh, Omar O., Gootenberg, Jonathan S.

Issue&Volume: 2022-11-24

Abstract: Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR–Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.

DOI: 10.1038/s41587-022-01527-4

Source: https://www.nature.com/articles/s41587-022-01527-4

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