Challenges and opportunities in adapting high-throughput functional assays for in vivo neuroscience.

Abstract

High-throughput functional assays, including CRISPR perturbations with single-cell readouts and massively parallel reporter assays, are redefining studies of gene regulation and function. These assays are effective in homogeneous cultured cells, where libraries can be efficiently scaled, delivered, and recovered. The brain, however, imposes steeper barriers. Viral packaging limits, sparse recovery, and restricted expression constrain throughput, while cellular heterogeneity, spatial architecture, and activity-dependent dynamics demand greater resolution. We review these challenges and emerging strategies for adapting high-throughput functional assays to in vivo functional genomics in neuroscience. We outline advances, obstacles, and a framework organized around two central challenges: (1) library delivery and signal recovery, and (2) cellular complexity. Overcoming these barriers is essential to unlocking how gene regulation shapes brain function and disease.