Genome-wide CRISPR screening in the organism has tremendous potential to answer long-standing questions of mammalian physiology and disease. However, bringing this powerful technology in vivo presents unique challenges, including delivering a genome-wide sgRNA library to the appropriate cell type, achieving sufficient coverage of the library, and selecting for the phenotype of interest. In this review, we highlight recent advances in sgRNA delivery, library design, and phenotypic readout that can help overcome these technical challenges and thereby bring high-throughput genetic dissection to an increasing number of tissues and questions. We are excited about the potential for ongoing innovation in these areas to ultimately enable genome-wide CRISPR screening in any cell type of interest in the organism, allowing for unprecedented investigation into diverse questions of mammalian physiology and disease.