CRISPR-mediated gap-based replacement enables site-specific DNA lesion insertion into long DNA and nucleosomes

Defined DNA lesions are essential substrates for dissecting DNA repair mechanisms, yet existing approaches remain limited in positional flexibility, lesion diversity, efficiency, or compatibility with long and chromatinized DNA. Here, we present CGRID (CRISPR-mediated Gap-based Replacement for Insertion of DNA damage), a programmable strategy for site-specific installation of DNA lesions into long DNA and nucleosomes. CGRID uses paired Cas9 nickases to generate two adjacent nicks, followed by gap formation and replacement with a synthetic lesion-containing oligonucleotide. This design enables easy and efficient insertion of diverse DNA lesions at user-defined positions without pre-engineered nicking sites or substrate redesign. Applied to long λ-DNA, CGRID supports efficient installation of single lesions and internal double-strand breaks at predefined positions, which are challenging to obtain using existing methods. These molecules enable high-throughput single-molecule analysis of diverse repair pathways including base excision repair (BER), nonhomologous end joining, and microhomology-mediated end joining without hydrodynamic force. CGRID further allows efficient site-specific lesion incorporation into nucleosomal DNA, enabling systematic repositioning of damage within a nucleosome. This shows that nucleosome context and lesion orientation strongly influence APE1 incision efficiency. Together, CGRID establishes a broadly applicable platform for programmable DNA damage engineering, bridging biochemical and single-molecule methodologies, and facilitates mechanistic studies of chromatin-dependent genome maintenance.