Biology subjects

Marin, D.

Publications and source records attributed to Marin, D..

7 recordsLinked to original sources

bioRxiv2026

Efficient base editing and development in human embryos without chromosomal alterations

Cas9-based tools enable the introduction of genetic lesions to investigate DNA repair outcomes and edit the genome at disease-relevant loci. DNA double-strand breaks (DSBs) induced by CRISPR/Cas9 result in frequent aneuploidy and large deletions, revealing a repair deficiency in early human embryos and limiting the clinical application of this technology. Here we evaluated the DNA repair outcomes of DNA nicks and mismatches introduced using base editors in human embryos at two targets, PCSK9 and HBG. Editing was efficient and, unlike Cas9-induced DSBs, did not result in either chromosomal abnormalities or large deletions. Small insertions or deletions after base editing were rare, and off-target activity was dependent on the guide RNA. Delivering the base editor as a protein at fertilization or at the pronuclear stage allowed normal development to the blastocyst stage and the derivation of edited stem cell lines. In stark contrast, introduction of the editor as RNA resulted in early embryo arrest. Our results demonstrated that, unlike DSBs, DNA nicks and mismatches are efficiently repaired in human embryos, allowing specific on-target changes without genotoxic consequences.

genetics↗

bioRxiv2025

DSB repair kinetics impair CRISPR-Cas9 editing in human embryos

Cas9 provides a powerful tool to interrogate DNA repair and to introduce targeted genetic modifications. However, a major challenge of Cas9-based editing in human embryos is the occurrence of chromosomal abnormalities caused by Cas9 cleavage. Furthermore, mosaicism - different genetic outcomes in different cells, prevent accurate genotyping using a single embryo biopsy. Through timed analysis of editing outcomes during the first cell cycle and timed inhibition of Cas9 using AcrIIA4, we show that most edits occur at least 12 hours post Cas9 injection and therefore after the first S-phase. This timing limits the ability to achieve uniform editing across cells. We found that segmental chromosomal abnormalities and the consequential loss of heterozygosity are common at Cas9 cleavage sites throughout the genome, including at MYBPC3 and at CCR5 loci, for which this has not previously been reported. Surprisingly, inhibiting Cas9 activity 8-12 hours before mitosis does not eliminate chromosomal aneuploidies. This suggests that double-strand break (DSB) repair in human embryos is exceedingly slow, with breaks remaining unrepaired for many hours. Thus, the timing of DSB induction and repair relative to the first S-phase and the first mitosis is intrinsically limiting to preventing mosaicism and maintaining genome stability in embryonic gene editing.

developmental biology↗

bioRxiv2022

DNA Double Strand Breaks cause chromosome loss through sister chromatid tethering in human embryos

Summary paragraphGenome editing by DNA double-strand breaks (DSB) is currently being investigated as a tool to treat or even prevent heritable diseases1. However, DNA repair mechanisms in the human embryo remain poorly understood and DSBs may result in chromosome loss 2,3. Here we provide evidence of whole and segmental chromosome loss in over one third of chromosomes 16, 17 and X targeted by CRISPR/Cas9-induced DNA DSB, including pericentromeric and mid-arm sites. Chromosomal changes were asymmetric relative to the Cas9 cut site: segmental losses occurred on both centric as well as acentric chromosome arms, while gains were exclusively found on acentric arms, suggesting that centromeres in broken chromosomes continued to mediate sister chromatid separation. Using this pattern of chromosomal errors, we were able to define new genomic coordinates of the active centromere on chromosome 16. Asymmetry was also found in the attrition of gDNA at the break site: attrition occurred centromeric of the DSB, while telomeric to the break, chromosomal ends were protected. Thus, spindle forces at centromeres and end tethering and protection at DSBs are antagonistic forces that interfere with accurate segregation of sister chromatids. Thereby, a single DSB is sufficient to result in the loss of a chromosome from the embryo. These results highlight the risks of aneuploidy in CRISPR/Cas9 genome editing, while also providing a mechanism for mitotically acquired aneuploidy caused by DNA breaks in human embryos.

developmental biology↗

bioRxiv2020

Generation of glucocorticoid resistant SARS-CoV-2 T-cells for adoptive cell therapy

Adoptive cell therapy with viral-specific T cells has been successfully used to treat life-threatening viral infections, supporting the application of this approach against COVID-19. We expanded SARS-CoV-2 T-cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observed that the choice of cytokines modulates the expansion, phenotype and hierarchy of antigenic recognition by SARS-CoV-2 T-cells. Culture with IL-2/4/7 but not other cytokine-driven conditions resulted in >1000 fold expansion in SARS-CoV-2 T-cells with a retained phenotype, function and hierarchy of antigenic recognition when compared to baseline (pre-expansion) samples. Expanded CTLs were directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T-cells could not be efficiently expanded from the peripheral blood of non-exposed controls. Since corticosteroids are used for the management of severe COVID-19, we developed an efficient strategy to inactivate the glucocorticoid receptor gene (NR3C1) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.

immunology↗

bioRxiv2020

Reading frame restoration at the EYS locus, and allele-specific chromosome removal after Cas9 cleavage in human embryos

The correction of disease-causing mutations in human embryos could reduce the burden of inherited genetic disorders in the fetus and newborn, and improve the efficiency of fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here we evaluate the repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frame-shift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. However, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, upon entry into mitosis, loss of one or both chromosomal arms. Thus, Cas9 allows for the modification of chromosomal content in human embryos in a targeted manner, which may be useful for the prevention of trisomies. HighlightsO_LICas9-mediated DSB induction and repair by end joining occurs within hours C_LIO_LIEnd joining provides an efficient way to restore reading frames without mosaicism C_LIO_LIUnrepaired DSBs persist through mitosis and result in frequent chromosome loss C_LI

genetics↗

bioRxiv2020

Inhibition of the av integrin-TGF-b axis improves natural killer cell function against glioblastoma stem cells

Glioblastoma, the most aggressive brain cancer, often recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. Here, we show that patient-derived GSCs, but not normal astrocytes, are highly sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. In contrast, single cell analysis of autologous, tissue infiltrating NK cells isolated from surgical samples of high-grade glioblastoma patient tumors using mass cytometry and single cell RNA sequencing revealed an abnormal phenotype associated with impaired lytic function compared with peripheral blood NK cells from GBM patients or healthy donors. This immunosuppression was attributed to an integrin-TGF-{beta} mechanism, activated by direct cell-cell contact between GSCs and NK cells. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-{beta} signaling, or with TGF-{beta} receptor 2 gene-edited NK cells prevented GSC-induced NK cell dysfunction and tumor growth. Collectively, our findings reveal a novel mechanism of NK cell immune evasion by GSCs and implicate the integrin-TGF-{beta} axis as a useful therapeutic target to eliminate GSCs in this devastating tumor.

immunology↗

bioRxiv2020

CIS checkpoint deletion enhances the fitness of cord blood derived natural killer cells transduced with a chimeric antigen receptor

Immune checkpoint therapy has produced remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible SH2-containing (CIS) protein, a key negative regulator of interleukin (IL)-15 signaling, with chimeric antigen receptor (CAR) engineering of natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell anti-tumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that combining CIS checkpoint deletion with CAR engineering promotes the metabolic fitness of NK cells in an otherwise suppressive tumor microenvironment. This approach, together with the prolonged survival afforded by CAR modification, represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.

immunology↗