Lymphocyte Biology Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;
Lymphocyte Biology Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Immunohorizons. 2021 Feb 23;5(2):117-132. doi: 10.4049/immunohorizons.2000082.
Clustered regularly interspaced short palindromic repeats (CRISPR)-based methods have revolutionized genome engineering and the study of gene-phenotype relationships. However, modifying cells of the innate immune system, especially macrophages, has been challenging because of cell pathology and low targeting efficiency resulting from nucleic acid activation of intracellular sensors. Likewise, lymphocytes of the adaptive immune system are difficult to modify using CRISPR-enhanced homology-directed repair because of inefficient or toxic delivery of donor templates using transient transfection methods. To overcome these challenges and limitations, we modified existing tools and developed three alternative methods for CRISPR-based genome editing using a hit-and-run transient expression strategy, together with a convenient system for promoting gene expression using CRISPRa. Overall, our CRISPR tools and strategies designed to tackle both murine and human immune cell genome engineering provide efficient alternatives to existing methods and have wide application not only in terms of hematopoietic cells but also other mammalian cell types of interest.
基于成簇规律间隔短回文重复序列（CRISPR）的方法已经彻底改变了基因组工程和基因-表型关系的研究。然而，由于细胞病理学和核酸激活细胞内传感器导致的靶向效率低，修饰固有免疫系统的细胞，特别是巨噬细胞，一直具有挑战性。同样，由于使用瞬时转染方法对供体模板进行低效或有毒的递送，适应性免疫系统的淋巴细胞也难以使用 CRISPR 增强的同源定向修复进行修饰。为了克服这些挑战和限制，我们修改了现有的工具，并开发了三种替代方法，用于使用命中和运行的瞬时表达策略进行基于 CRISPR 的基因组编辑，同时还开发了一种使用 CRISPRa 促进基因表达的便捷系统。总的来说，我们设计的用于解决小鼠和人类免疫细胞基因组工程的 CRISPR 工具和策略为现有方法提供了高效的替代方案，不仅在造血细胞方面具有广泛的应用，而且在其他感兴趣的哺乳动物细胞类型方面也具有广泛的应用。
