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.
Copyright © 2021 The Authors.