Yehia, G. | bioRxiv Science

bioRxiv2026

Cardiomyocyte-derived Wnt5a drives doxorubicin-induced cardiomyopathy by amplifying cellular senescence

Doxorubicin (DOX) is an effective anthracycline chemotherapeutic agent, but its use is limited by cardiotoxicity that can progress to cardiomyopathy and heart failure. Cellular senescence contributes to DOX-induced cardiac injury, yet the upstream signals that initiate and propagate senescence in the injured heart remain unclear. Here, we identify Wnt5a, a non-canonical Wnt ligand, as a mediator of anthracycline cardiomyopathy. WNT5A was increased in serum from cancer patients receiving anthracycline therapy and in a pathologic human cardiomyocyte population in the context of DOX-induced cardiomyopathy. In mouse hearts, DOX induced early cardiomyocyte-enriched Wnt5a expression before overt cardiac dysfunction. Cardiomyocyte-specific Wnt5a deletion attenuated DOX-induced cardiac dysfunction, fibrosis and senescence marker induction, whereas recombinant Wnt5a and cardiomyocyte-targeted Wnt5a overexpression were sufficient to promote cardiomyocyte senescence and cardiac dysfunction. Mechanistically, DOX activated a Wnt5a-Fzd2 feed-forward axis that amplified Wnt5a expression in cardiomyocytes and propagated senescence to neighboring fibroblasts. Genetic disruption of this pathway in cardiomyocytes, fibroblasts or senescent cells reduced DOX-induced cardiomyopathy. Pharmacological inhibition of Wnt5a signaling with secreted frizzled-related protein 5 suppressed DOX-induced cardiac injury without compromising the anticancer efficacy of DOX. These findings identify Wnt5a-Fzd2 signaling as a senescence-amplifying mechanism in anthracycline cardiomyopathy and suggest a therapeutic strategy to mitigate DOX cardiotoxicity.

cell biology↗

bioRxiv2026

dCas allele sequestration (das-CRISPR): A Versatile New Method to Achieve Monoallelic Gene Editing in Mouse Embryos and in cell culture.

CRISPR-Cas9 technology is a powerful tool extensively used for genome editing in mouse and many other species. Streptococcus pyogenes Cas9 efficiently cuts both alleles in mouse zygotes leaving many edited embryos without a functional protein that might be needed to sustain development, to survive postnatally or to reproduce, thus complicating its overwhelmingly advantageous use in making gene modifications. About 25% of mouse genes are essential for embryonic development and another 7% are necessary for fertility, thus for these genes it is desirable to maintain a functional allele to establish viable lines from CRISPR-Cas9 edited mouse embryos. However, exclusive monoallelic editing is challenging to achieve with current CRISPR methods. Controlling the activity of Cas9 in genome editing is an ongoing research field focused on developing new methods to curtail its damage caused by excess of on-target and off-target editing. In this study we describe a novel and a simple method, we termed das-CRISPR, for dCas allele sequestration in combination with CRISPR system, that allows monoallelic editing of targeted allele in mouse and in cultured cell lines. This method incorporates the use of a nuclease deficient deadCas9 (dCas9) present at higher levels than an active Cas9, both complexed with the same single guide RNA (sgRNA) sequence. We showed the delivery of the two proteins as ribonucleoprotein complexes (RNP) into mouse zygotes leads to the generation of viable and fertile mice carrying lethal mutations in an essential gene. We found that greater amounts of dCas9 RNPs bind and protect a target site while the lower amount of functional Cas9 RNPs accessed the unoccupied target site resulting in higher frequency of monoallelic gene editing, compared to using just Cas9 alone. We also showed this method can mitigate and control the activity of Cas9 in mouse NIH3T3 cells in culture to achieve monoallelic editing. This method is a versatile approach to controlling excessive Cas9 activity on-target and off-target both in vitro and in vivo.

molecular biology↗