Project Summary/Abstract Systemic lupus erythematosus (SLE) is a debilitating systemic autoimmune disease that results from the breakdown in B cell tolerance and ultimately the generation of pathogenic autoreactive antibodies. Multiple factors contribute to SLE including genetic predisposition and environmental influences. Our Emory Autoimmunity Center of Excellence has characterized a pathogenic lineage of extrafollicular (EF) B cells in Black SLE subjects in the US South and demonstrated that these EF B cells harbor a unique epigenetic and transcriptional profile. Importantly, EF B cells are predisposed to differentiate into antibody secreting plasma cells (ASC), are highly expanded in SLE disease flares, and harbor autoreactive B cell receptors. Despite these studies, we still do not know which genes underpin EF B cells in SLE and contribute to the secretion of autoreactive antibodies. This Pilot Project will use sophisticated CRISPR/Cas9 gain and loss-of-function screens in primary SLE and healthy B cells to unravel the gene networks and interactions that promote the differentiation of naïve and EF B cells in SLE. Importantly, we will assay two distinct stages of B cell differentiation to answer the following questions. Do antigen inexperienced SLE naive B cells have distinct differentiation requirements from healthy? How do EF B cells differ from classical memory cells in their requirements to form ASC? Can over expression of EF genes in naive B cells drive more rapid ASC differentiation? Do driver genes in SLE also promote ASC formation in healthy B cells or do they require other SLE specific changes, such as cofactors or epigenetic remodeling? To answer these questions, we propose two aims. In Aim 1, we will define the genes required for SLE B cells to form ASC using loss-of-function screens. Importantly we will also test which genes require or synergize with TLR7 signaling to promote ASC formation. In Aim 2, we will identify driver genes of SLE B cell differentiation to ASC using gain-of-function screens. Here, genes upregulated in different EF SLE B cell compartments will be over expressed in antigen-inexperienced naïve B cells to determine if they are sufficient to enhanced ASC formation. Finally, we will perform the screen while simultaneously overexpressing TBET to identify genes that collaborate with this known driver of EF B cell fate and autoimmunity. The outcomes of this Pilot Project will derive the genes required for SLE B cells to form ASC and establish a genome engineering platform that can be leveraged for future mechanistic studies of human autoimmunity.