Droplet-based functional CRISPR screening of cell-cell interactions by SPEAC-seq

Cell-cell interactions are essential for the function and contextual regulation of biological tissues. We present a platform for high-throughput microfluidics-supported genetic screening of functional regulators of cell-cell interactions. Systematic perturbation of encapsulated associated cells followed by sequencing (SPEAC-seq) combines genome-wide CRISPR libraries, cell coculture in droplets and microfluidic droplet sorting based on functional read-outs determined by fluorescent reporter circuits to enable the unbiased discovery of interaction regulators. This technique overcomes limitations of traditional methods for characterization of cell-cell communication, which require a priori knowledge of cellular interactions, are highly engineered and lack functional read-outs. As an example of this technique, we describe the investigation of neuroinflammatory intercellular communication between microglia and astrocytes, using genome-wide CRISPR-Cas9 inactivation libraries and fluorescent reporters of NF-κB activation. This approach enabled the discovery of thousands of microglial regulators of astrocyte NF-κB activation important for the control of central nervous system inflammation. Importantly, SPEAC-seq can be adapted to different cell types, screening modalities, cell functions and physiological contexts, only limited by the ability to fluorescently report cell functions and by droplet cultivation conditions. Performing genome-wide screening takes less than 2 weeks and requires microfluidics capabilities. Thus, SPEAC-seq enables the large-scale investigation of cell-cell interactions.