Using CRISPR/cas9 in primary natural killer cells to study antibody dependent cellular cytotoxicity signaling.

Abstract

Abstract In contrast to conventional natural killer (NK) cells, adaptive NK cells lack expression of the Fc receptor γ chain and transcription factor PLZF. A higher proportion of adaptive NK cells is correlated with protection from malaria. Adaptive NK cells have shown enhanced degranulation in response to opsonized targets, as measured by CD107A and interferon-γ (IFN-γ) expression. We hypothesize that people with a higher frequency of adaptive NK cells are protected from malaria because of the enhanced degranulation of adaptive NK cells. However, it is unclear why adaptive NK cells degranulate better than conventional NK cells. To answer this question, we developed a CRISPR/cas9 protocol to ablate genes associated with the antibody-dependent cellular cytotoxicity (ADCC) signaling pathway and evaluated the effect of these ablations on NK cell degranulation. Individual gene targets included the Fc receptor γ chain, CD3ζ, Syk, ZAP70, and PLZF. Combinations of these targets were also ablated. After ablating gene(s) of interest, mutant NK cells were incubated with an opsonized target and evaluated five hours later for CD107A and IFN-γ expression. As a control, we found that the combined ablation of the Fc receptor γ and CD3ζ chain severely diminishes NK cell degranulation. Our results from other gene targets will be shown. Furthermore, details of our CRISPR/cas9 method will be shared. The method is the first to effectively ablate multiple primary NK cell targets using the Amaxa 4D nucleofector. On average, we obtained 82.8% protein ablation across all targets. Overall, this work will give insight into why adaptive NK cells function better at ADCC and help the field effectively ablate genes in primary NK cells.