Chromosome conformation capture (3C)-derived methods have become an indispensable tool in the study of gene regulation. The three-dimensional contacts they are able to assay 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. 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. ### Competing Interest Statement V.I.R. and J.S. are inventors on a related patent application covering CAD-C (PCT application filed 2024). NIH Common Fund, https://ror.org/001d55x84, 1DP2GM150021 Irma T. Hirschl Trust, https://ror.org/01yaqvf46, Career Scientist Award Rita Allen Foundation, https://ror.org/0515k5w36, Scholar Award Stavros Niarchos Foundation, https://ror.org/0210rze73, Institute for Global Infectious Disease Research at Rockefeller University Grant Robertson Technology Development Fund at Rockefeller University Boehringer Ingelheim (Germany), https://ror.org/00q32j219, PhD Fellowship to JS U.S. National Science Foundation, https://ror.org/021nxhr62, GRFP to LAW International Human Frontier Science Program Organization, https://ror.org/02ebx7v45, Postdoctoral Cross-Disciplinary Fellowship to AO Natural Sciences and Engineering Research Council of Canada, Postgraduate fellowship to HC, Postgraduate fellowship to JLY

Magdalene College is deeply saddened to announce the death of Professor Sir John Gurdon FRS, who served as Master of the College from 1995 to 2002.

Researchers have devised a way to visualize molecules that are very rare, very small, or hard to produce naturally, including viruses.

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Specialized magnetic beads that bind target proteins to a cryogenic electron microscopy grid make it possible to study the structure of protein complexes from dilute samples.