小柯机器人

美国约翰·霍普金斯大学医学院Taekjip Ha团队利用DISCOVER-Seq+提高体内CRISPR脱靶检测的灵敏度。2023年4月6日，《自然—方法学》杂志在线发表了这项成果。

研究人员证明，对DNA依赖性蛋白激酶催化亚基的抑制会使修复蛋白MRE11在CRISPR-Cas靶向位点聚集，从而能够使用染色质免疫沉淀法和测序法将脱靶位点与MRE11结合的位置进行高灵敏度的映射。这种技术被称为DISCOVER-Seq+，与以前的方法相比，在永生细胞系、原生人类细胞和小鼠中发现了多达五倍的CRISPR脱靶点。研究人员证明了适用于原代人T细胞中癌症导向的转基因T细胞受体的体外敲入和小鼠体内心血管风险基因PCSK9的腺病毒敲除。因此，DISCOVER-Seq+是迄今为止发现体内非靶标基因组编辑的最敏感方法。

据了解，在病人来源的细胞和动物模型中发现脱靶CRISPR-Cas活动对基因组编辑应用至关重要，但目前表现出低敏感性。

附：英文原文

Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

Author: Zou, Roger S., Liu, Yang, Gaido, Oscar E. Reyes, Konig, Maximilian F., Mog, Brian J., Shen, Leo L., Aviles-Vazquez, Franklin, Marin-Gonzalez, Alberto, Ha, Taekjip

Issue&Volume: 2023-04-06

Abstract: Discovery of off-target CRISPR–Cas activity in patient-derived cells and animal models is crucial for genome editing applications, but currently exhibits low sensitivity. We demonstrate that inhibition of DNA-dependent protein kinase catalytic subunit accumulates the repair protein MRE11 at CRISPR–Cas-targeted sites, enabling high-sensitivity mapping of off-target sites to positions of MRE11 binding using chromatin immunoprecipitation followed by sequencing. This technique, termed DISCOVER-Seq+, discovered up to fivefold more CRISPR off-target sites in immortalized cell lines, primary human cells and mice compared with previous methods. We demonstrate applicability to ex vivo knock-in of a cancer-directed transgenic T cell receptor in primary human T cells and in vivo adenovirus knock-out of cardiovascular risk gene PCSK9 in mice. Thus, DISCOVER-Seq+ is, to our knowledge, the most sensitive method to-date for discovering off-target genome editing in vivo.

DOI: 10.1038/s41592-023-01840-z

Source: https://www.nature.com/articles/s41592-023-01840-z

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