Understanding gene function is critical for developing therapeutic strategies to target disease. Common approaches to understanding gene function in a systematic and unbiased way include loss-of-function and gain-of-function genomic screening. Some of these rely on artificially increasing the copy number of gene transcripts using cDNA expression libraries. Others interrogate endogenous protein expression through genetic loss-of-function approaches such as siRNA screening. Over the past two decades, targeted approaches that reduce the endogenous expression level of genes or proteins have been developed that facilitate a much better understanding of genes in the context of the living cell. Technologies have been developed that enable precise modification of the genome rather than reduction at the transcript level. These include zinc finger nucleases, TALENs and the CRISPR/Cas9 technology. The use of these systems in large-scale high-throughput screening is an emerging field and we herein highlight recommendations for such applications.