BPS2026 – Light-activated CRISPR for on-demand perturbation of chromatin insulators

The ability to manipulate chromatin in real-time could transform our understanding of genomic organization and processes. CRISPR-Cas9 systems are frequently used to precisely target genomic regions; however, they lack the temporal control necessary to interrogate nuclear processes at physiological timescales. By strategically incorporating light-sensitive non-canonical nucleotides into the seed region of a guide RNA, we transformed catalytically inactive dCas9 into a programmable, light-activated DNA-binding switch, which we term “seed-gated CRISPR”. This approach achieves synchronized and highly specific binding at sub-minute timescales in vitro and in cells. Single-molecule force spectroscopy revealed that CTCF, a chromatin loop-anchoring factor, dynamically unwraps and rewraps DNA, transiently exposing its motif. Leveraging seed-gated CRISPR, we exploited this conformational exploration to displace CTCF from select binding sites. This strategy enables rapid, targeted, and on-demand chromatin manipulation, opening new avenues for studying genome organization and dynamic nuclear processes at timescales previously inaccessible.