Understanding how cis-regulatory function is encoded in DNA sequence using massively parallel reporter assays and designed sequences

•New methods offer high-throughput testing of designed cis-regulatory elements.•These assays are used to validate genomic predictions of cis-regulatory function.•Designed reporter libraries offer improved statistical power for hypothesis testing.

Genome-scale methods have identified thousands of candidate cis-regulatory elements (CREs), but methods to directly assay the regulatory function of these elements on a comparably large scale have not been available. The inability to directly test and perturb the regulatory activity of large numbers of DNA sequences has hindered efforts to discover how cis-regulatory function is encoded in genomic sequence. Recently developed massively parallel reporter gene assays combine next generation sequencing with high-throughput oligonucleotide synthesis to offer the capacity to test and mutationally perturb thousands of specifically chosen or designed cis-regulatory sequences in a single experiment. These assays are the basis of recent studies that include large-scale functional validation of genomic CREs, exhaustive mutational analyses of individual regulatory sequences, and tests of large libraries of synthetic CREs. The results demonstrate how massively parallel reporter assays with libraries of designed sequences provide the statistical power required to address previously intractable questions about cis-regulatory function.