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使用CRISPR-Cas9核糖蛋白复合体介导的基因剥离来研究小鼠CD4 T细胞分化

Jillian K Perry¹, Pamela L Schwartzberg¹, Dominic P Golec²

¹Laboratory of Immune System Biology, NIAID, NIH.

Journal of visualized experiments : JoVE

|July 7, 2025

概括

这项研究介绍了CRISPR-Cas9协议用于小鼠T细胞中的基因淘汰. 这种方法使得在T细胞分化过程中能够有效地评估基因功能,而无需依赖昂贵的动物模型.

科学领域:

免疫学免疫学免疫学
分子生物学分子生物学
基因编辑基因编辑

背景情况:

聚类正规间隔短平行体重复 (CRISPR) -Cas9技术为功能性研究促进了初级T细胞的基因向.
在T细胞中进行初步基因功能测试时生成淘汰 (KO) 鼠标菌株通常具有成本限制,并且可用性有限.
商业上可用的资源,如预定导向RNA (gRNA),可方便地生成基因向的原始T细胞,用于激活和分化研究.

研究的目的:

用CRISPR-Cas9核核蛋白复合体 (RNPs) 来生成基因向的小鼠原始CD4T细胞的协议.
为了在体外 CD4 T 细胞分化中有效评估基因功能.
提供一种方法,绕过与传统基因向动物模型相关的限制.

主要方法:

从小鼠的二次淋巴体器官中分离原始的CD4 T细胞.
在激活之前,用CRISPR-Cas9-gRNA复合体对分离的T细胞进行核感染,以实现基因淘汰 (KO).
试验室极化协议以诱导分化到Th1,Th2,Th17和Treg系.

主要成果:

通过使用核感染传递的CRISPR-Cas9 RNP,有效地生成原始CD4 T细胞.
成功评估CD4 T细胞分化途径中的基因功能.
在各种下游应用中证明CD4或CD8T细胞的适应性,包括体外激活和体内采用转移研究.

结论:

基于核感染的CRISPR-Cas9协议提供了一种简化和高效的方法,用于初级T细胞中基因KO.
这种方法克服了KO动物模型和T细胞前激活输送方法的局限性,使得基因功能分析具有稳健性.
该协议有助于在T细胞分化和激活研究中对众多基因进行常规评估,促进免疫学研究.

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