Project Summary This project will develop a new tool, SpliceTag, for conditional knock-in/knockout and demonstrate its utility. SpliceTag uses highly cell type specific alternative exons as a vehicle for carrying protein and RNA tags for conditional labeling, or premature stop codons for protein knockout. It can be introduced in an intron of the target gene through CRISPR/Cas9 induced homology directed repair. The SpliceTag exon will then be spliced in mRNA labeling the protein in the desired cell type. In Aim 1 we will create a mouse line in which we use a SpliceTag in Rpl22 to label ribosomes with ALFA peptide in photoreceptor cells. The photoreceptor specific SpliceTag is based on Ttc8 microexon 2a which we have shown in the past to be highly specific to photoreceptor cells. To demonstrate the utility of our approach we will use the Rpl22 SpliceTag line to analyze translational efficiency in photoreceptor cells and how translation is controlled during the diurnal cycle. In Aim 2 we will determine the feasibility of using SpliceTag to label proteins in epithelial cell and inner retina neurons. We will also determine if it can incorporate commonly used tags, SNAP and CBP/Strep, that are significantly larger than the Ttc8 exon 2a on which SpliceTag is based. Since SpliceTag does not rely on recombinases it can enable experimental designs where recombinase and floxed alleles are used for genetic manipulations in parallel with SpliceTag, including knockouts in cell types other than the cell type in which the protein is being labeled.