Generating mammalian knock-out cell lines to investigate mitochondrial protein complex assembly

The development of easy-to-use gene-editing approaches has revolutionized the study of mitochondrial protein complex assembly in mammalian cells. Once the domain of classical cell biology models such Saccharomyces cerevisiae, human knockout cell lines lacking expression of a specific protein can be made at low cost and in as little as two to three weeks. In this chapter we outline our approach to generation of knockouts in commonly used transformed laboratory cell lines, with a view toward their use in the study of mitochondrial respiratory chain complex assembly and mitochondrial biology. Common pitfalls and caveats are discussed along with recommendations on how to validate a knockout cell line through sequencing of genomic edits and stable complementation to exclude the influence of off-target effects and enable further studies of protein function.