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

Soroczynski, J.; Westcott, L. A.; Zuo, W.; Ou, A.; Canaj, H.; Hickling, J.; Yeung, J. L.; Konishi, H. A.; Campbell, E. B.; Whelan, C.; Balacco, J.; Formenti, G.; Risca, V. I.

doi:10.64898/2025.12.22.695891

bioRxiv · 10.64898/2025.12.22.695891

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

Soroczynski, J.·Westcott, L. A.·Zuo, W.·Ou, A.·Canaj, H.·Hickling, J.·Yeung, J. L.·Konishi, H. A.·Campbell, E. B.·Whelan, C.·Balacco, J.·Formenti, G.·Risca, V. I.

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Abstract

Chromosome conformation capture (3C)-derived methods have become an indispensable tool in the study of gene regulation. The three-dimensional contacts probed by 3C methods 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 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. Leveraging the ligation-competent DNA ends produced by CAD cleavage, we show that CAD-C is compatible with a highly streamlined, repair-free protocol and produces multi-step CADwalks, consecutive ligations between nucleosomal or sub-nucleosomal fragments. With these walks, we probe local chromatin fiber folding contacts, nucleosomal and sub-nucleosomal footprints, and long-range nuclear organization regimes in human cell lines. CAD-C is an efficient, robust chromatin structure assay that can span sub-nucleosomal to chromosomal length scales in a single experiment.

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Soroczynski, J., Westcott, L. A., Zuo, W., Ou, A., Canaj, H., Hickling, J., Yeung, J. L., Konishi, H. A., Campbell, E. B., Whelan, C., Balacco, J., Formenti, G., Risca, V. I.. 2025-12-22. CAD-C: An engineered nuclease enables repair-free in situ proximity ligation and nucleosome-resolution chromosome walks in human cells. https://doi.org/10.64898/2025.12.22.695891

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