Enzyme tethering for in situ epigenomics

Genome-wide mapping of factors that bind, package, replicate and transcribe DNA continues to be challenging despite rapid advances in DNA sequencing technologies. For nearly two decades, chromatin immunoprecipitation with sequencing (ChIP–seq) has dominated epigenomic mapping applications; however, alternative methods for epigenomic mapping have recently begun to replace ChIP–seq. The most popular alternatives use enzymes that modify, cleave or transpose DNA, using freely diffusing enzymes for chromatin accessibility mapping, or tethered ones to specific chromatin proteins or modifications for epigenomic profiling. Unlike ChIP–seq, which solubilizes chromatin before immunoprecipitation, enzyme-tethering epigenomic methods maintain cells or nuclei intact during DNA modification, cleavage or transposition. Although the basic in vivo and in situ enzyme-tethering technologies were introduced more than 20 years ago, they are especially well suited for current epigenomic challenges, including single-cell and spatial applications and both DNA-based and RNA-based time-resolved epigenomic mapping. In this Primer, we discuss DNA modification-based (DamID), cleavage-based (ChIC and CUT&RUN) and transposase-based (CUT&Tag and RT&Tag) tethered-enzyme profiling methods, and describe protocols for data processing, quality control and data analysis used by the epigenomics community. We anticipate that advances in enzyme engineering and automation will make enzyme-tethering methods increasingly attractive for both large and small epigenomic profiling projects.