Mutations in trans-acting factors such as transcription factors, chromatin regulators, and DNA repair genes may cause widespread transcriptomic changes or altered genome-wide mutation patterns leading to a variety of phenotypes with varying impact on human health. Functional consequences of these mutations are difficult to systematically evaluate on a large scale due to the many challenges of trans studies. In this dissertation, I focus on two types of trans¬-acting factors: DNA repair genes, which control genome-wide mutation signatures, and transcriptional regulators, which impact genome-wide expression patterns. First, I present a study in which we used an unbiased genome-wide scan for regulators of repeat expansion propensity to identify the mismatch repair protein MSH3 as a strong trans-acting factor affecting germline mutation patterns in recombinant inbred mice. We found that inherited variants in and near Msh3 result in variable mutation patterns that are most pronounced at long tetranucleotide repeats. Importantly, we also demonstrate a potential evolutionary tradeoff in which elevated Msh3 leads to increased repeat expansions whereas Msh3 deficiency results in a higher rate of short insertions and deletions. Second, I introduce a novel trans-eQTL detection method, xQTL, which is based on a biologically plausible mixture model of target gene effects sizes and results in increased power compared to traditional trans-eQTL analysis approaches. We applied xQTL to whole brain RNA-sequencing data from a cohort of outbred rats and identified 45 trans-eQTL candidates. For example, we identified a strong candidate trans-eQTL locus overlapping Neurod4, a key neuronal transcriptional factor, which xQTL estimate to regulate thousands of target genes. Importantly, this study also highlights key technical considerations regarding treatment of technical covariates when performing trans-eQTL detection. Last, I introduce scBE-seq which combines a pooled, high-precision genome editing strategy with single-cell sequencing assays to simultaneously interrogate the effects of hundreds of variants affecting trans-acting factors. Overall, these works furthered our understanding of the molecular effects of genetic variation on trans-acting factors and extended our toolkit for systematically studying their potential impact on complex phenotypes.