Michael A. Wheeler^1,2†, Iain C. Clark^1,3†, Hong-Gyun Lee^1†, Zhaorong Li^1,2, Mathias Linnerbauer¹, Joseph M. Rone¹, Manon Blain⁴, Camilo Faust Akl¹, Gavin Piester^1,5, Federico Giovannoni¹, Marc Charabati¹, Joon-Hyuk Lee¹, Yoon-Chul Kye^1,6, Joshua Choi^1,6, Liliana M. Sanmarco¹, Lena Srun¹, Elizabeth N. Chung¹, Lucas E. Flausino¹, Brian M. Andersen^1,7, Veit Rothhammer^1,8, Hiroshi Yano⁹, Tomer Illouz¹, Stephanie E. J. Zandee¹⁰, Carolin Daniel^11,12,13, David Artis^9,14, Marco Prinz^15,16,17, Adam R. Abate^18,19, Vijay K. Kuchroo^1,2,6, Jack P. Antel⁴, Alexandre Prat¹⁰, Francisco J. Quintana^1,2*

¹Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA.
²Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
³Department of Bioengineering, College of Engineering, California Institute for Quantitative Biosciences, University of California Berkeley, Berkeley, CA 94720, USA.
⁴Neuroimmunology Unit,Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal, QC H3A 2B4, Canada.
⁵Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY 14642, USA.
⁶Evergrande Center for Immunologic Diseases, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA 02115, USA.
⁷Department of Neurology, Jamaica Plain Veterans Affairs Hospital, Harvard Medical School, Boston, MA 02130, USA.
⁸Department of Neurology, University Hospital Erlangen, Friedrich-AlexanderUniversity Erlangen-Nuernberg, Erlangen, Germany.
⁹Jill Roberts Institute for Research in Inflammatory Bowel Disease, Joan and Sanford I. Weill Department of Medicine, Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY 10021, USA.
¹⁰Neuroimmunology Research Lab, Centre de Recherche du Centre Hospitalier de l'Universitéde Montréal, Montreal, QC H2X 0A9, Canada.
¹¹Type 1 Diabetes Immunology, Helmholtz Diabetes Center at Helmholtz Zentrum München, 80939 Munich, Germany.
¹²Deutsches Zentrum für Diabetesforschung, 85764 Munich-Neuherberg, Germany.
¹³Division of Clinical Pharmacology, Department of Medicine IV, Ludwig-Maximilians-Universität München, 80337 Munich, Germany.
¹⁴Friedman Center for Nutrition and Inflammation, Joan and Sanford I. Weill Department of Medicine, Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, NewYork, NY 10021, USA.
¹⁵Institute of Neuropathology, University of Freiburg, D-79106 Freiburg, Germany.
¹⁶Signaling Research Centres BIOSS and CIBSS, University of Freiburg, D-79106 Freiburg, Germany.
¹⁷Center for Basics in Neuro Modulation, Faculty of Medicine, University of Freiburg, D-79106 Freiburg, Germany.
¹⁸Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California Institute for Quantitative Biosciences, San Francisco, CA 94158, USA.
¹⁹Chan Zuckerberg Biohub, San Francisco, CA, USA.

^*Corresponding author: Francisco J. Quintana
^†These authors contributed equally to this work.

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.