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

Abstract

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