A key goal for cell biological analyses is to assess the phenotypes that result from eliminating a target gene. For the past 25 years, the predominant strategy utilized in human tissue culture cells has been RNAi-mediated protein depletion. However, RNAi suffers well-documented off-target effects as well as incomplete and reversible protein depletion. The implementation of CRISPR/Cas9-based DNA cleavage has revolutionized the capacity to conduct functional studies in human cells.