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Homodimeric cyanine dyes are DNA intercalators that display a large enhancement of fluorescence emission when bound to double-stranded DNA. However, other different interaction modes are possible, such as H-type molecular aggregates of the dye, templated by the nucleic acid. In this paper, we study in depth the formation of nonfluorescent H-aggregates of the cyanine homodimer YOYO-3 with two different DNA templates using absorption and both steady-state and time-resolved fluorescence spectroscopy. First, a nonfluorescent YOYO-3 H-aggregate complex was found to form in single-stranded polycytidine chains, resulting in the appearance of a new absorption band at approximately 500 nm. The specific interaction of cytosine bases suggests the involvement of the C-rich i-motif in facilitating the formation of the H-aggregate complex. Second, the interaction of YOYO-3 with double-stranded poly(A·T) tracts also led to the appearance of a new absorption band at approximately 500 nm, and hence of a different type of H-aggregate. We found that the aggregate is formed mainly in double-stranded regions with consecutive adenine bases in the same strand (and thymine bases in the complementary strand). These poly(A·T) tracts provide narrow minor grooves and enhanced electrostatic negative potential to promote the aggregation of the negatively charged cyanine. As the YOYO-3 H-aggregates are nonfluorescent, our results provide an important basis to quantitatively understand the fluorescence emission of this cyanine dye in the presence of DNA strands.

Summary Efficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22) translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1) using end-joining DNA processing factors involved in repair mechanisms, or (2) ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3) all-in-one plasmid or ribonucleoprotein complex-based approaches. We report that the generation of targeted t(11;22) is significantly increased by using a combination of ribonucleoprotein complexes and ssODNs. The CRISPR/Cas9-mediated generation of targeted t(11;22) in human stem cells opens up new avenues in modeling Ewing sarcoma., Graphical Abstract, Highlights • T All-in-one vector has improved function by an extra NLS and coexpression of elements • Usage of RNP and ssODN recreates efficiently human chromosomal translocations • The Ewing sarcoma t(11;22) translocation is unstable in hMSCs, In this article, Rodriguez-Perales and colleagues show an efficient CRISPR approach for recreating cancer-associated chromosome translocations in human stem cells. They generate the Ewing sarcoma t(11;22) translocation in human mesenchymal and induced pluripotent stem cells using RNPs and ssODNs. The generation of targeted translocations in human stem cells opens up new avenues in modeling Ewing sarcoma and human neoplasias.