Optimization of CRISPR-Cas9 Mediated Knockout Protocol in Primary Human Myeloid Cells

Abstract Myeloid cells are an important player in the immune system, and form first line of defense against pathogens, such as Mycobacterium tuberculosis (Mtb), causative pathogen for (TB) disease. They include macrophages, a target Mtbinfection, dendritic (DCs), which present Mtb-derived antigens to prime activate T cells. In order investigate myeloid-specific host factors involved response we sought optimize protocol genetically edit primary human monocyte-derived DCs macrophages subsequent infection experiments with Mtb. We built on prior published by Hiatt, et al. using nucleofection ribonucleoproteins (RNP), Cas9 recombinant protein complexed multiplexed guide RNA (gRNA) molecules. tested reaction volume, RNP concentration, rest time, programs. found that molar ratio Cas9:gRNA was most efficient at 1:2 1.8μM final concentration combination 2hr time after before adding differentiation supplement. Lonza program EA100 proved be optimal recovered conclusion, optimized robust editing culturing edited macrophages. This opens opportunities interrogation candidate TB risk genes Fumarylacetoacetate hydrolase (FAH). These can now used mechanistic studies Mtbinfection define mechanisms pathogenesis.