CAD-C: An engineered nuclease enables repair-free in situ proximity ligation and nucleosome-resolution chromosome walks in human cells II

The three-dimensional contacts can probe depend strongly on the properties of the enzyme used to fragment chromatin prior to proximity-driven ligation. Micrococcal nuclease (MNase), used in Micro-C, increases resolution at the expense of low ligation efficiency and the need for extensive enzyme titration.

To overcome these limitations, we engineered a highly active, TEV protease-activatable caspase-activated DNase (CAD) to enable an efficient, low-sequence-bias, and high-resolution proximity ligation assay we call CAD-C. CAD-C was successful on the first attempt for each human cell line tested and the resulting datasets capture loops, TADs, compartments, and stripes similarly to Micro-C. However, compared to Micro-C and Hi-C, CAD-C shows enhanced sensitivity for promoter-enhancer loops.