Biology subjects

Arvidsson, E.

Publications and source records attributed to Arvidsson, E..

3 recordsLinked to original sources

bioRxiv2026

CRISPRa-mediated activation of Tfeb in a fibril-induced alpha-synuclein aggregation model

Accumulation of misfolded alpha-synuclein (a-syn) is a key hallmark of Parkinson's disease (PD), making it a key target for novel therapeutic strategies. Here, we use a previously described screening assay to select multiple efficient single guide RNA (sgRNA) combinations targeting transcription factor EB (TFEB) for CRISPR-activation (CRISPRa) using a nuclease-deficient Staphylococcus aureus Cas9 coupled to a truncated VP64-p53-Rta (VPR) activator complex. Selected sgRNA are applied in a rat neuroblastoma in-vitro model of fibril-induced a-syn aggregation with the goal of inducing autophagy and reduce protein aggregation. We successfully induce a-syn aggregation in this model and observe a stagnation in phosphorylated a-syn (p-a-syn) compared to non-phosphorylated a-syn across treatment groups. While we achieve robust expression of Tfeb in the exogenous screening assay and at endogenous mRNA level, we did not observe proportional increase in protein expression. Additionally, we do not observe any additive effects of CRISPRa induction combined with media treatments inducing Tfeb nuclear translocation. Taken together, these findings illustrate difficulties in epigenetic induction of Tfeb using CRISPRa and highlights the need for additional studies exploring this application.

molecular biology↗

bioRxiv2023

A systematic screening assay identifies efficient small guide RNAs for CRISPR activation

CRISPR-mediated gene activation (CRISPRa) encompasses a growing field of biotechnological approaches with exciting implications for gene therapy. However, there is a lack of experimental validation tools for selecting efficient sgRNAs for downstream applications. Here, we present a screening assay capable of identifying efficient single- and double sgRNAs through fluorescence quantification in vitro. In addition, we provide a tailored Golden Gate cloning workflow for streamlined incorporation of selected sgRNA candidates into lentiviral (LVs) or adeno-associated vectors (AAVs). The overall workflow was validated using therapeutically relevant genes for neurodegenerative diseases, such as Tfeb, Adam17, and Sirt1. The most efficient sgRNAs also demonstrated activation of endogenous gene expression at mRNA and protein levels. Further proof- of-principle assays using Tfeb indicated that gene activation was accompanied by increased levels of Lc3b. This data demonstrates the potential of the screening assay to identify functionally efficient sgRNA candidates across multiple genes along with streamlined cloning of viral vectors and may assist in accelerating future developments of CRISPRa-focused applications.

molecular biology↗

bioRxiv2022

Inducing synthetic lethality for selective targeting of acute myeloid leukemia cells harboring STAG2 mutations

Targeted therapies exploiting selective vulnerabilities of malignant cells are highly desired for clinical applications. The cohesin protein complex comprises of RAD21, SMC3, SMC1A as well as a fourth subunit that consists of either STAG1 or STAG2 and is essential for proper chromosomal segregation during mitosis. STAG2 loss-of-function mutations are recurrent driver events in acute myeloid leukemia (AML) and appear relatively early during leukemogenesis. Studies in cell lines have shown that STAG2 deficient cells are uniquely vulnerable to STAG1 perturbation, and this vulnerability could thus be exploited to selectively eliminate STAG2 null AML cells. Here we show that partial perturbation of STAG1 is well tolerated by normal human hematopoietic stem cells and does not affect their functionality. By contrast, STAG1 knockdown is lethal to STAG2 null human HSCs by inducing major mitotic defects. Moreover, STAG1 knockdown induced synthetic lethality in primary human AML cells harboring a STAG2 mutation and completely abrogated leukemia progression in xenograft models. Overall, our study provides proof-of-concept demonstration of a synthetic lethal approach to selectively target primary human cancer cells with STAG2 mutations

cancer biology↗