Development of a platform for CRISPR/CAS9 screening and engineering of iPSC-derived human microglia-like cells to validate neuroinflammation drug targets in Alzheimer's disease Rebecca S. Mathew1, Hong Sun1, Galya Vassileva1, Philip W. Garrett-Engele1, Sophia Bardehle2, Chris Mirescu2, Keith Tanis3, Yi Yang4, Ilona Kariv4, Matthew Kennedy2, and Joel Klappenbach1 1Merck & Co., Inc., Genetics & Pharmacogenomics, Boston, MA, USA; 2Merck & Co., Inc., Neuroscience, Boston, MA, USA; 3Merck & Co., Inc., Genetics & Pharmacogenomics, West Point, PA, USA; 4Merck Pharmacology, Boston, MA, USA Microglia are the innate immune cells of the brain and play a critical role in neurological disorders, including Alzheimer's disease (AD). Genome-wide association studies (GWAS) have identified several genes that are preferentially expressed by microglia and are associated with increased risk of developing late-onset AD, such as triggering receptor expressed on myeloid cells 2 (TREM2), myeloid cell surface antigen CD33 (CD33),and inositol polyphosphate-5-phosphatase (INPP5D). Designing reverse genetic experiments to ascertain the function of microglial ADassociated genes is hindered by the inability to obtain and manipulate postmortem tissue from AD patients. Human-induced pluripotent stem cell (hiPSC)-derived microglia cells represent a novel strategy to examine the relationship between genetic risk factors and late-onset AD. Recently, several groups have independently demonstrated that patient-derived hiPSCs can be differentiated into human microglia in vitro by providing cues that mimic the environment present in the developing embryo. Using iPSC-derived hematopoietic progenitor cells, we replicated the protocol published by Abud et al. (Neuron 94, 278-293, 2017) to generate induced microglial-like cells (“iMGLs”) and confirmed relevant phenotypes by wholetranscriptome, flow cytometry and immunocytochemical analysis. Functional analysis of iMGLs reveals that they secrete cytokines in response to inflammatory stimuli and CNS substrates, including Aβ fibrils. The ability to precisely introduce genetic disease-associated mutations in hiPSC-derived microglia cells using CRISPR/Cas9 technology will help define the contribution and function of genes associated with late-onset AD. We present a method to facilitate highgene activation and knockout studies of gene function via chemically inducible Cas9 and dCas9-fusion proteins.
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