Screens for genes that orchestrate neural circuit formation in mammals have been hindered by practical constraints of germline mutagenesis. To overcome these limitations, we combined RNA-seq with somatic CRISPR mutagenesis to study synapse development in the mouse retina. Here synapses occur between cellular layers, forming two multilayered neuropils. The outer neuropil, the outer plexiform layer (OPL), contains synapses made by rod and cone photoreceptor axons on rod and cone bipolar dendrites, respectively. We used RNA-seq to identify selectively expressed genes encoding cell surface and secreted proteins and CRISPR-Cas9 electroporation with cell-specific promoters to assess their roles in OPL development. Among the genes identified in this way are Wnt5a and Wnt5b. They are produced by rod bipolars and activate a non-canonical signaling pathway in rods to regulate early OPL patterning. The approach we use here can be applied to other parts of the brain.

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from "vanishingly rare" (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs "miRNAs"). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3' overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.