Socioeconomic factors and discrimination play major roles in variable cancer treatment outcomes across individuals from different ancestral backgrounds. Tumors from patients of different ancestry groups also have divergent patterns of somatic and germline alterations. This led us to hypothesize that these molecular differences may also contribute to the variable treatment outcomes observed in the clinic. We hypothesized that the ancestry of cell lines would impact their genetic dependencies. To test this hypothesis, we leveraged The Cancer Dependency Map (DepMap) which has performed genome-wide CRISPR screens across >1,000 cell lines and >30 cancer types. We first leveraged variant calls from WES/WGS to infer cell line ancestry, then correlated these ancestries with DepMap gene dependency scores. This analysis was underpowered to detect differences in cell lines of African, American, and South Asian descent, since cell lines from these ancestry groups are poorly represented in DepMap, and in cancer research models in general. We were, however, able to detect 71 gene dependencies that were associated with either European or East Asian ancestry. Since different ancestry groups have divergent patterns of germline alterations, we reasoned that specific germline alterations may result in ancestry-associated dependencies. Surprisingly, we identified cis-QTLs for >75% of the ancestry-associated genes. We originally hypothesized that these variants would alter the function of the encoded protein, but instead found that these variants mapped to the targeting sequences of the sgRNA. This suggests that ancestry-associated mismatches within sgRNA targeting sequences can preclude Cas9-mediated genome editing. To understand this problem systematically, we mapped the germline variants that were catalogued in gnomAD to multiple genome-wide CRISPR libraries. The fraction of affected genes differed from library to library and was typically between 2-5%, but we found that individuals of African descent were consistently more affected by this problem across all CRISPR libraries. This is important because it suggests that cell lines of African descent have a higher rate of false negatives in all CRISPR-based experiments. In total, we identified a subset of genes that have ancestry-associated dependency profiles. Most of these genes are the result of ancestry-associated mismatches within the sgRNA targeting sequences. However, many of these genes are not, and require further investigation to understand the influence of ancestry on these genetic dependencies. Citation Format: Sean Alexander Misek, Aaron Fultineer, Jeremie Kalfon, Javad Noorbakhsh, Isabella Boyle, Joshua Dempster, Lia Petronio, Katherine Huang, James McFarland, Rameen Beroukhim, Jesse Boehm. Ancestry bias in CRISPR guide design impedes discovery of genetic dependencies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2173.