CRISPR genome editing of human dendritic cells (treatments of knockout dendritic cells)

Dendritic cells (DCs) are central regulators of both innate and adaptive immunity and play roles in processes ranging from antitumor immunity to host-microbiome communication at mucosal surfaces. It remains difficult, however, to genetically manipulate human DCs, limiting our ability to probe how DCs elicit specific immune responses and to deploy DCs for immunotherapy. Here, we develop a CRISPR/Cas9 genome editing method for human monocyte-derived DCs (moDCs) with a median knockout efficiency of >93% across >300 genes. Using this method, we perform arrayed genetic screens in moDCs, identifying mechanisms by which human immune cells tune responses to lipopolysaccharides (LPSs) from the human microbiome. In addition, we reveal donor-specific responses to LPSs, underscoring the importance of assessing immune phenotypes in donor-derived cells, and identify genes that control this specificity, highlighting the potential of our method to pick apart genetic determinants of inter-individual variation in immune responses. Treatment of human monocyte-derived dendritic cells with knockouts of TLR4, LY96, CD14, or RPE65 (neutral gene control) with LPS from E. coli or B. theta or PBS (negative control) for 2 hours. 2 replicate treatments for each stimulus.