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

Abstract

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.