Tu149 - Single-cell CRISPRi Screen in Human Macrophages Identifies Immune Dysregulation in Crohn’s Disease

Elizabeth Aslinger – Icahn School of Medicine at Mount S; Christopher Tastad – Icahn School of Medicine at Mount S; Kyle Gettler – Icahn School of Medicine at Mount S; Michelle Bao – Icahn School of Medicine at Mount S; Diana Paguay – Icahn School of Medicine at Mount S; Colleen Chasteau – Icahn School of Medicine at Mount S; Jake Herb – Icahn School of Medicine at Mount S; Ksenija Sabic – Icahn School of Medicine at Mount S; Ling-shiang Chuang – Icahn School of Medicine at Mount S; Judy Cho – Icahn School of Medicine at Mount S

Abstract Text: Common and rare genetic variant Crohn’s alleles are enriched in loci containing genes expressed in myeloid cells, with many high effect genes being loss-of-function, notably NOD2. We employed CRISPRi screens and single-cell transcriptomics (Perturb-seq) to assess 41 IBD candidate genes, within 500kb of a Crohn’s- or IBD-associated SNP and 11 genes with known macrophage differentiation (such as CSF1R, SPI1 and PPARA). Following CRISPRi transduction, cells were cultured under hypoxia for 7 days with MDP, the NOD2-specific peptidoglycan agonist. Single cell sequencing demonstrated a median knockdown efficiency of 73% (median of 148 cells) per target. Among most differentially expressed genes of the integrated library, we observed two core, correlated perturbation clusters. The larger cluster contained numerous genes involved in MDP response, notably, NOD2, SNX20 (adjacent to NOD2), and RIPK2; the smaller cluster included PAF1, CSF1R, PPARA, CEBPB and ATG16L1. Complementary analysis using non-negative matrix factorization to dissect cell type from state confirmed the two core perturbation patterns. PPARA-targeted cells demonstrated downregulation of CSF1R transcript and the PI3K/Akt pathway. Some targets markedly altered mitochondrial expression and validation of three genes, STK11, CEBPB, and NRBP1, that upregulated the mitochondrial genes expression in our screen was performed by CRISPR knockout. The STK11 and NRBP1 knockouts both decreased mitochondrial ROS and favored glycolysis; CEBPB also decreased mitochondrial ROS, and both CEBPB and NRBP1 decreased mitochondrial mass. Given the central role of macrophage differentiation and cell state in gut hypoxia and with chronic microbial stimulation, these Perturb-Seq studies highlight mitochondrial function in Crohn’s pathogenesis.