小柯机器人

德国慕尼黑大学Oliver T. Keppler、Manuel Albanese等研究人员合作实现人类静息CD4⁺ T细胞的快速、高效基因编辑。2021年12月23日，国际知名学术期刊《自然—方法学》在线发表了这一成果。

通过使用优化的细胞培养和Cas9引导的RNA核糖核蛋白复合物的核染条件，研究人员报告了一种快速、高效的人类静息CD4⁺ T细胞基因编辑方法。多达六个基因，包括人类免疫缺陷病毒（HIV）依赖性和限制因子，被单独或同时敲除，并进行了功能鉴定。此外，研究人员展示了将双链DNA供体模板敲入不同的内源性位点，从而能够在单细胞分辨率下研究细胞和病毒成分的生理性相互作用。总之，这种技术可以改善HIV生物学和静息CD4⁺ T细胞免疫功能的分子和功能特征。

据介绍，CD4⁺T细胞是适应性和先天性免疫反应的核心媒介，在体内构成了HIV的一个主要储存者。对静息的人类CD4⁺T细胞的详细调查被排除在外，因为缺乏有效的基因操作方法，这限制了人们对HIV复制的理解，也限制了治疗方法的寻找。

附：英文原文

Title: Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses

Author: Albanese, Manuel, Ruhle, Adrian, Mittermaier, Jennifer, Mejas-Prez, Ernesto, Gapp, Madeleine, Linder, Andreas, Schmacke, Niklas A., Hofmann, Katharina, Hennrich, Alexandru A., Levy, David N., Humpe, Andreas, Conzelmann, Karl-Klaus, Hornung, Veit, Fackler, Oliver T., Keppler, Oliver T.

Issue&Volume: 2021-12-23

Abstract: CD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9–guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.

DOI: 10.1038/s41592-021-01328-8

Source: https://www.nature.com/articles/s41592-021-01328-8

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