Genetic manipulations with a robust and predictable outcome are critical to investigate gene function, as well as for therapeutic genome engineering. For many years, knockdown approaches and reagents including RNA interference and antisense oligonucleotides dominated functional studies; however, with the advent of precise genome editing technologies, CRISPR-based knockout systems have become the state-of-the-art tools for such studies. These technologies have helped decipher the role of thousands of genes in development and disease. Their use has also revealed how limited our understanding of genotype–phenotype relationships is. The recent discovery that certain mutations can trigger the transcriptional modulation of other genes, a phenomenon called transcriptional adaptation, has provided an additional explanation for the contradicting phenotypes observed in knockdown versus knockout models and increased awareness about the use of each of these approaches. In this review, we first cover the strengths and limitations of different gene perturbation strategies. Then we highlight the diverse ways in which the genotype–phenotype relationship can be discordant between these different strategies. Finally, we review the genetic robustness mechanisms that can lead to such discrepancies, paying special attention to the recently discovered phenomenon of transcriptional adaptation.

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