Cyanine-modified ssODNs enhance CRISPR-Cas9 HDR in stem cell embryo models via chromatin and chemical modulation

While CRISPR–Cas9 has transformed biomedical research, precise genome editing via homology-directed repair (HDR) remains limited by low efficiency and cytotoxicity, particularly in sensitive systems. Here, we show that simple 5′ cyanine modifications of single-stranded oligodeoxynucleotides (ssODNs) markedly enhance HDR efficiency across human stem cell models, providing a 2–3-fold survival advantage. This approach enables in situ HDR in 3D human embryo models (blastoids), achieving up to 30% HDR—a 2.5–3-fold improvement over unmodified ssODNs—without compromising viability or developmental potential. Edited blastoids recapitulate key implantation features, including epiblast outgrowths surrounded by hypoblasts and trophoblasts. Mechanistically, enhanced HDR is associated with upregulation of HDR-related genes, including linker histone H1.0, which binds 5′Cy5-ssODNs and promotes HDR. Additionally, 5′Cy5 increases stability and nuclear availability of ssODNs. Molecular dynamics and free energy analyses suggest that 5′Cy5 promotes compact, circular-like conformations, validated by atomic force microscopy, and enhances donor–target duplex stability. Together, these findings establish cyanine-modified ssODNs as a simple, biocompatible strategy to improve precise genome editing. Homology-directed repair (HDR) is constrained by low efficiency and cytotoxicity, particularly in sensitive systems. Here, the authors show that 5′ cyanine-modified ssODN templates enhance CRISPR-Cas9 HDR efficiency and improve cell survival in human stem cells and embryo models.