The completion of the Human Genome Project in 2003 marked the beginning of the functional genomics era, allowing research to extend beyond identifying genes and instead focus on how these genes and their products function and interact within biological systems. Since then, one of the most pivotal high throughput technologies developed was the use of CRISPR/Cas9 gene editing to perform pooled perturbation screens to identify genetic regulators of a given phenotype of interest. While these CRISPR-based screens were originally limited to growth-based phenotypes, additional efforts have allowed for more advanced phenotypes to be studied. Here, we have expanded upon these advancements to perform increasingly complex screens. First, we performed a FACS-based, genome-wide CRISPR/Cas9 knockout screen for regulators of recombinant adeno-associated virus (rAAV) production. rAAV-mediated gene therapies are a well-established and effective treatment, yet the cost and labor associated with manufacturing rAAV vectors can be prohibitive. To this end, we identified genes that can be manipulated in producer cells to increase rAAV yield. Next, we developed a fluorescence-based protein cleavage reporter that can be coupled with large-scale FACS-based screens to study proteolysis at scale. Protein processing is an essential post-translational modification involved in a variety of cellular processes, and cleavage has been implicated in many diseases, including Huntington’s disease. We harnessed this cleavage reporter to perform both an overexpression and a knockout screen for modifiers of a key cleavage event in Huntington’s disease pathogenesis. Through these screens we identified a gene that indirectly regulates this cleavage event and generated a list of other genes worthy of further investigation. Lastly, we developed a protocol to combine RNA FISH with in situ sequencing and performed optical pooled screens in a myotonic dystrophy type 1 (DM1) patient fibroblast line. These screens identified genes that may potentially modulate the toxic RNA foci seen in DM1. Taken together, we have demonstrated the power of pooled perturbation screening to improve current therapeutics and to identify genes that may prove useful therapeutic targets for neurodegenerative diseases.