Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9

Abstract

Myeloid cells play critical and diverse roles in mammalian physiology, including tissue development and repair, innate defense against pathogens, and generation of adaptive immunity. As cells that show prolonged recruitment to sites of injury or pathology, myeloid cells represent therapeutic targets for a broad range of diseases. However, few approaches have been developed for gene editing of these cell types, likely owing to their sensitivity to foreign genetic material or virus-based manipulation. Here we describe optimized strategies for gene disruption in primary myeloid cells of human and murine origin. Using nucleofection-based delivery of Cas9-ribonuclear proteins (RNPs), we achieved near population-level genetic knockout of single and multiple targets in a range of cell types without selection or enrichment. Importantly, we show that cellular fitness and response to immunological stimuli is not significantly impacted by the gene editing process. This provides a significant advance in the study of myeloid cell biology, thus enabling pathway discovery and drug target validation across species in the field of innate immunity.

Using Gene Editing Approaches to Fine-Tune the Immune System

“…Despite the low efficiency of viral and non-viral delivery methods, several NK cells can be edited to enhance their persistence, cytotoxicity, and tumor targeting (117). Dendritic cells (DCs) play a critical role in T-cell response instructions, with triple knockout established as proof of concept (118). A similar approach is used to target the costimulatory molecule CD40, whose disruption significantly inhibits T-cell activation, thus reducing graft damage and prolonging graft survival (88).…”

CRISPR-based functional genomics in human dendritic cells

“…To enable introduction of specific knockouts in moDCs, we developed a non-viral genome editing strategy based on electroporation of in vitro-assembled Cas9-sgRNA complexes (Cas9 ribonucleoprotein particles, RNPs), an approach that has been validated in other immune cell types (Freund et al, 2020;Hiatt et al, 2020;Riggan et al, 2020;Roth et al, 2018;Schumann et al, 2015). Briefly, our strategy entails isolating monocytes from human donor blood, differentiating them into moDCs in the presence of GM-CSF and IL-4, and electroporating these moDCs with Cas9 RNPs to induce double-strand breaks at the targeted locus ( Figure 1a).…”

“…As a consequence, DC biology is generally studied in mouse models, but mice and humans differ in many aspects of both innate and adaptive immunity, including innate immune receptor repertoires, responses to immune ligands such as lipopolysaccharide (LPS), and developmental pathways of adaptive immune cells (Pulendran and Davis, 2020). One way to address this challenge is to knock out genes in DC precursor populations such as monocytes or stem cells, followed by differentiation into DCs (Freund et al, 2020;Hiatt et al, 2020;Laustsen et al, 2018). These methods, however, require independent differentiation of each knockout population and as a result are susceptible to batch effects and poorly suited for genetic screens.…”

CRISPR-based functional genomics in human dendritic cells

“…As a consequence, DC biology is generally studied in mouse models, but mice and humans differ in many aspects of both innate and adaptive immunity, including innate immune receptor repertoires, responses to immune ligands such as lipopolysaccharide (LPS), and developmental pathways of adaptive immune cells ( Pulendran and Davis, 2020 ). One way to address this challenge is to knock out genes in DC precursor populations such as monocytes or stem cells, followed by differentiation into DCs ( Freund et al, 2020 ; Hiatt et al, 2020 ; Laustsen et al, 2018 ). These methods, however, require independent differentiation of each knockout population and as a result are susceptible to batch effects and poorly suited for genetic screens.…”