CRISPR-based functional genomics in human dendritic cells

Marco Jost, Amy N. Jacobson, Jeffrey A. Hussmann, Giana Cirolia,  Michael A. Fischbach,  Jonathan S. Weissman
Source: BioResearch Open Access
Publication Date: (2020)
Issue: 1: 1-76
Research Area:
Immunotherapy / Hematology
Cells used in publication:
Dendritic cell (NHDC), human
Species: human
Tissue Origin: blood
4D-Nucleofector® 96-well Systems

The final electroporation condition (solution P3, pulse code DJ-108, 4 · 105 cells
electroporated with 10 pmol Cas9 and 25 pmol sgRNA) was obtained through iterative grid searches of different conditions. In the process, several other conditions were also found to yield good results, including nucleofection solution P1 with pulse code CB-128 (Figure 1 – Figure Supplement 3a). In initial experiments, we also used solution P1 with pulse code CB-150 (Figure 1c, Figure 1 – Figure Supplement 1). Reducing the amount of Cas9 RNP led to a reduction in editing efficiency.


Dendritic cells (DCs) regulate processes ranging from antitumor and antiviral immunity to host-microbe communication at mucosal surfaces. It remains difficult, however, to genetically manipulate human DCs, limiting our ability to probe how DCs elicit specific immune responses. Here, we develop a CRISPR/Cas9 genome editing method for human monocyte-derived DCs (moDCs) that mediates knockouts with a median efficiency of >93% across >300 genes. Using this method, we perform genetic screens in moDCs, identifying mechanisms by which DCs tune responses to lipopolysaccharides from the human microbiome. In addition, we reveal donor-specific responses to lipopolysaccharides, 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 pinpoint determinants of inter-individual variation in immune responses. Our work sets the stage for a systematic dissection of the immune signaling at the host-microbiome interface and for targeted engineering of DCs for neoantigen vaccination.