Massively parallel single-cell CRISPR screen for development of novel microglia targeting immunotherapies in Alzheimer’s Disease

Prof. Dr. Ido Amit

• A03

Dr. Hadas Keren-Shaul

• A03

Project Summary

Immune signaling has been implicated in Alzheimer’s disease (AD) development and progression, but it remains unclear which cells, genes, and pathways are activated during the pathology. Microglia are the central nervous system (CNS) intrinsic mechanism of protection, and we have shown that dedicated sensory receptors are critically involved in the transition from microglia homeostatic state to a disease-associated microglia (DAM) protective state upon CNS damage. Analysis of microglia revealed that DAM diff erentiation is a sequential two-step process involving multiple sensory receptors, transcriptional circuits, and effector genes. However, the functional role of the different DAM pathways and checkpoints have not been elucidated. The lack of a robust and widely applicable technology for high-throughput invivo screening of multiple microglial checkpoint candidates has been a major stumbling block in identifying essential DAM-related pathways which promote immune-protective and nurturing activities of microglia and prevent long-term microglial and DAM exhaustion. Here, we aim to overcome this challenge and shed light on the missing key knowledge of the role of microglia in AD by developing a microglia-specific massively parallel single-cell genomic technology coupled
with CRISPR pooled screens (miCRISPR-seq) (Aim 1). By simultaneously introducing multiple genetic perturbations in microglia-specific Cas9-expressing AD mice, we will screen the most relevant DAM specific genes to identify essential DAM regulators and checkpoints (Aim 2). Single-cell RNA sequencing (MARS-seq) combined with direct sequencing of the guide RNAs and the single-cell transcriptome, will allow us to discover novel targets that activate or dampen the DAM activity and restore protective immune activity in AD brains and to decipher the therapeutic and mechanistic effects of boosting or dampening the DAM state (Aim 3). Our miCRISPR-screen will help to define novel therapeutic targets for preclinical validation.