Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins

Abstract

Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloidlineage cells have remained largely genetically intractable. We present a method for delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically-edited cells that retain critical markers of both myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection more than fifty-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate development of novel myeloid cellular therapies.

M. tuberculosisinfection of human iPSDM reveals complex membrane dynamics during xenophagy evasion

“…Although this system provides a wealth of cells for investigations and likely recapitulates the interactions that occur between Mtb and recruited macrophages, their ability to reflect the tissue-resident alveolar macrophages, which show different responses to Mtb infection ( Pisu et al, 2020 ), is unclear and donor-to-donor variability can confound results. Additionally, these human monocyte-derived macrophages are not genetically tractable – they are difficult to transfect and CRISPR/Cas9-mediated gene knockout has only been reported once ( Hiatt et al, 2020 preprint). One alternative is the differentiation of the immortalised monocytic cell line THP-1 with phorbol esters to produce macrophages.…”

CRISPR-based functional genomics in human dendritic cells

“…To enable introduction of specific knockouts in human 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

“…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.…”