Evolutionary innovation emerges through the interplay of genetic novelty, genomic architecture and evolutionary constraint. Nature Reviews Genetics presents a Focus issue that explores the mechanisms that generate, integrate and shape biological diversity.

In this Tools of the Trade article, Yujie Chen describes CHARM, a single-cell four-omics sequencing method that profiles genome conformation, chromatin accessibility, histone modifications and gene expression.

In this Tools of the Trade article, Dmitry Kretov and Daniel Cifuentes describe RBPscan (RNA-binding protein specificity and contextual analysis via nucleotide editing), a method that measures the strength of in vivo interactions between RNA binding proteins and their RNA targets.

Sarah Kocher reflects on two influential 2016 studies in the ruff that demonstrated how a large genomic inversion links variation across multiple traits into a single ‘supergene’.

In this Comment, Patricia Wittkopp revisits assumptions that cis-regulatory mutations are weakly pleiotropic, ultimately calling for more data to assess the extent to which they are less pleiotropic and deleterious than trans-regulatory mutations.

In this Comment, Cedric Feschotte highlights evidence from diverse lineages showing that transposable elements are key drivers of evolutionary innovation, repeatedly introducing regulatory and coding sequences that reconfigure genome function.