Optimized Nonviral Gene Disruption in Primary Murine and Human Myeloid Cells

Abstract

Genetically engineered myeloid cells such as monocytes, macrophages, and dendritic cells have broad applications in basic and translational research. Their central roles in innate and adaptive immunity make them attractive as putative therapeutic cell products. However, efficient gene editing of primary myeloid cells presents unique challenges owing to their sensitivity to foreign nucleic acids and poor editing efficiencies using current methodologies (Hornung et al., Science 314:994-997, 2006; Coch et al., PLoS One 8:e71057, 2013; Bartok and Hartmann, Immunity 53:54-77, 2020; Hartmann, Adv Immunol 133:121-169, 2017; Bobadilla et al., Gene Ther 20:514-520, 2013; Schlee and Hartmann, Nat Rev Immunol 16:566-580, 2016; Leyva et al., BMC Biotechnol 11:13, 2011). This chapter describes nonviral CRISPR-mediated gene knockout in primary human and murine monocytes as well as monocyte-derived or bone marrow-derived macrophages and dendritic cells. Electroporation-mediated delivery of recombinant Cas9 complexed with synthetic guide RNAs can be applied for population-level disruption of single or multiple gene targets.

Keywords: Bone marrow; CRISPR; Cas9; Cell biology; Dendritic cells; Electroporation; Gene editing; Genetic engineering; Human; Immunology; Innate immunity; Knockout; Macrophages; Monocytes; Mouse; Murine; Myeloid cells; RNP.