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Functionally dissecting the transcriptional regulatory network of the innate immune response using CRISPR-effectors

Specific Aims

Response to pathogen detection is probably one of the most fundamental processes in life. Innate immune cells have the responsibility to detect and classify the pathogen followed rapidly by a transcriptional program specific to the identified pathogen type. Dysregulation of components involved in the pathway at any level has been shown to result in severe pathologies, ranging from thwarted response to infection, autoimmune disease and cancer.  Studies by our group and many others have found that mammalian regulatory networks are rich and highly redundant. Although critically important in evaluating the result of a cis- or trans-regulatory mutation, we are still unable to accurately predict the result of a defective trans-activator or the impact of the loss of a cis-regulatory element on the rest of the regulatory network. Here we propose to apply an innovative CRISPR-effector method to fully probe the regulatory network that orchestrates the response of dendritic cells to pathogen-specific stimuli.

The last couple of years have seen a vertiginous pace in the development of CRISPR/Cas9-based approaches for genome editing and of targeting of effectors (repressors and activators) to specific genomic loci. We have established this technology in our laboratory with the specific goal of dissecting transcriptional regulatory networks controlling both development and cellular response to external stimuli. In our recent published work, we showed the use of both a repressive (KRAB) and activator (VP64) Cas9 systems to successfully destabilize the pluripotency gene regulatory network and were able to activate differentiation factors through targeting of involved cis-regulatory elements. More recently, we have established a mouse model that stably expresses an inducible effector Cas9-KRAB system and have further developed a lysine-specific histone demethylase 1 (LSD1) Cas9 effector system. The Cas9-LSD1 system specifically decommissions enhancer elements and has no effect when targeted to promoter regions. The efficacy and specificity of the CRISPR effector (CRISPRe) system make it a powerful tool to dissect regulatory elements. We propose to establish this system as the experimental backbone of transcriptional network deconvolution in primary dendritic cells. Specifically, we propose to use CRISPRe to dissect the innate immune transcriptional network in dendritic cell stimulated with LPS that we have previously described. We will then use our dCas9-effectors to address the several outstanding questions.

Aim 1

Define the mode of operation of stage-specific transcription factors (TFs) in the response of DCs using Cas9-repressors

We’ll use a murine cas9 effector system to repress key transcription factors in bone marrow derived DCs response to immunogens. 

Aim 2

Characterize the functional role of enhancer elements in DC maturation using Cas9-effector mediated enhancer decommissioning

We’ll rely on a cas9 effector system that specifically targets enhancer regions to characterize the function of key cis-regulatory regions in regulating response to immunogens in DCs.