Biology subjects

Kagermazova, L.

Publications and source records attributed to Kagermazova, L..

4 recordsLinked to original sources

bioRxiv2025

Selective depletion of cancer cells with extrachromosomal DNA via lentiviral infection

Dear Editor Extrachromosomal DNA (ecDNA), a major focal oncogene amplification mode found across cancer, has recently regained attention as an emerging cancer hallmark1,2, with a pervasive presence across cancers3,4. With technical advancements such as high-coverage sequencing and live-cell genome imaging, we can now investigate ecDNAs behaviors and functions 5-7. However, we still lack an understanding of how to eliminate ecDNA. We observed depletion of cells containing ecDNA during lentiviral but not transposon-based transduction while we sought to investigate the mechanism of ecDNA behavior. This discovery may provide critical information on utilizing a lentiviral system in emerging ecDNA research. Additionally, this observation suggests specific sensitivities for cells with ecDNA.

cancer biology↗

bioRxiv2023

Engineered transcription-associated Cas9 targeting in eukaryotic cells

DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT). Engineered TraCT is enabled in eukaryotic yeast or human cells when suboptimal PAM interactions limit basal activity and when one or more nascent RNA substrates are still tethered to the actively transcribed target DNA in cis. Using yeast, we further show that this phenomenon can be applied for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9s targeting activity at specific DNA sites may be engineered without modifying Cas9s core domains and guide RNA components or their expression levels. More broadly, it establishes co-transcriptional RNA binding as a cis-acting mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotic cells.

bioengineering↗

bioRxiv2023

Integrative Multi-omic Profiling of Two Human Decedents Receiving Pig Heart Xenografts Reveals Strong Perturbations in Early Immune-Cell and Cellular Metabolism Responses.

BackgroundRecent advances in xenotransplantation in living and decedent humans using pig xenografts have laid promising groundwork towards future emergency use and first in human trials. Major obstacles remain though, including a lack of knowledge of the genetic incompatibilities between pig donors and human recipients which may led to harmful immune responses against the xenograft or dysregulation of normal physiology. In 2022 two pig heart xenografts were transplanted into two brain-dead human decedents with a minimized immunosuppression regime, primarily to evaluate onset of hyper-acute antibody mediated rejection and sustained xenograft function over 3 days. MethodsWe performed multi-omic profiling to assess the dynamic interactions between the pig and human genomes in the first two pig heart-xenografts transplants into human decedents. To assess global and specific biological changes that may correlate with immune-related outcomes and xenograft function, we generated transcriptomic, lipidomic, proteomic and metabolomics datasets, across blood and tissue samples collected every 6 hours over the 3-day procedures. ResultsSingle-cell datasets in the 3-day pig xenograft-decedent models show dynamic immune activation processes. We observe specific scRNA-seq, snRNA-seq and geospatial transcriptomic changes of early immune-activation leading to pronounced downstream T-cell activity and hallmarks of early antibody mediated rejection (AbMR) and/or ischemia reperfusion injury (IRI) in the first xenograft recipient. Using longitudinal multiomic integrative analyses from blood in addition to antigen presentation pathway enrichment, we also observe in the first xeno-heart recipient significant cellular metabolism and liver damage pathway changes that correlate with profound physiological dysfunction whereas, these signals are not present in the other xenograft recipient. ConclusionsSingle-cell and multiomics approaches reveal fundamental insights into early molecular immune responses indicative of IRI and/or early AbMR in the first human decedent, which was not evident in the conventional histological evaluations.

systems biology↗

bioRxiv2018

CRISPR/Cas9-based Editing of a Sensitive Transcriptional Regulatory Element to Achieve Cell Type-Specific Knockdown of the NEMO Scaffold Protein

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-{kappa}B signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter A) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support canonical NF-{kappa}B signaling in response to treatment with tumor necrosis factor (TNF). Nevertheless, non-canonical NF-{kappa}B signaling is intact in these NEMO-deficient cells. Expression of ectopic NEMO in the edited cells restores downstream NF-{kappa}B signaling in response to TNF. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter A) in the human SNU-423 liver cancer cell line. We have also used homology directed repair (HDR) to fix the promoter B element in a 293T cell clone. Overall, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-{kappa}B signaling are discussed.

molecular biology↗