Use of CRISPR/Cas9 Gene Editing Methods to Investigate the Mechanism of Trem2-Dependent Gene Expression in Macrophages

Abstract

Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is a surface receptor expressed in macrophages during tissue injury. This receptor plays a role in driving phagocytosis and dampening inflammation. Because of this, it plays a large part in diseases such as Alzheimer’s disease, liver fibrosis, and metabolic syndrome. Each of these diseases all have a population of TREM2-expressing macrophages that does not exist in healthy tissue. However, the exact pathway in which TREM2 is involved in these diseases is rather unknown. Macrophage gene expression is regulated by a variety of transcription factors such as ATF3 and TFEB. These transcription factors have been suggested to be involved in some of the disease processes mentioned above by RNA-seq or ChIP-seq experiments.The research question we addressed was how these two transcription factors directly affectvtranscription in macrophages, specifically in the TREM2 pathway. CRISPR/Cas9 gene editing was used to generate loss of function alleles for each transcription factor. RNA-seq was then used to compare gene expression to define thegene-specific transcriptional roles of each factor and determine whether they play roles downstream of TREM2 signaling.Results showed that Atf3 knockout had very few genes upregulated or downregulated in the RNA seq compared to Trem2 knockout. Tfeb, on the other hand, had 13 genes in common with Trem2 knockout that were expressed lower than the control and 10 genes in common expressed higher than the control. The Tfeb knockout hadno difference in Trem2 expression between the knockout population and control, further providing evidence that Tfeb is located downstream of Trem2. Because Trem2 levels stayed consistent in the Tfeb KO, it is likely thatsome of the effects of Trem2 on the macrophage disease population genes are directly mediated through Tfeb.