The CRISPR/Cas9 system is transforming many biomedical disciplines, including cancer research. Through its flexible programmability and efficiency to induce DNA double strand breaks it has become straightforward to introduce cancer mutations into cells in vitro and/or in vivo. However, not all mutations contribute equally to tumorigenesis and distinguishing essential mutations for tumor growth and survival from biologically inert mutations is cumbersome. Here we present a method to screen for the functional relevance of mutations in high throughput in established cancer cell lines. We employ the CRISPR/Cas9 system to probe cancer vulnerabilities in a colorectal carcinoma cell line in an attempt to identify novel cancer driver mutations. We designed 100 high quality sgRNAs that are able to specifically cleave mutations present in the colorectal carcinoma cell line RKO. An all-in-one lentiviral library harboring these sgRNAs was then generated and used in a pooled screen to probe possible growth dependencies on these mutations. Genomic DNA at different time points were collected, the sgRNA cassettes were PCR amplified, purified and sgRNA counts were quantified by means of deep sequencing. The analysis revealed two sgRNAs targeting the same mutation (UTP14A: S99delS) to be depleted over time in RKO cells. Validation and characterization confirmed that the inactivation of this mutation impairs cell growth, nominating UTP14A: S99delS as a putative driver mutation in RKO cells. Overall, our approach demonstrates that the CRISPR/Cas9 system is a powerful tool to functionally dissect cancer mutations at large-scale.