Biology subjects

Lundin, A.

Publications and source records attributed to Lundin, A..

5 recordsLinked to original sources

bioRxiv2025

Artemis Regulates Homology-Independent Prime Editing (PRINS) for Enhanced Genomic Insertions

Nuclease-based prime editing (PEn) offers enhanced genomic insertion efficiency compared to nickase-based prime editors, but its reliance on double-strand break (DSB) repair leads to complex and often unpredictable on-target indel distributions. PRINS editing, a PEn variant utilizing springRNAs, uniquely relies on non-homologous end joining (NHEJ) for insertions, providing an insertion-only strategy ideal for functional protein tagging or serine integrase landing pad insertions, yet it suffers from inherent imprecision. Here, we identify the DNA repair factor Artemis (DCLRE1C) as a key regulator of PEn-generated indel profiles, particularly in springRNA-mediated PRINS editing. Through a targeted genetic screen, we show that the absence of Artemis significantly shifts indel distributions away from deletions and shorter truncations towards longer, functionally acceptable insertions. Our data indicates that Artemis cleaves PEn-generated 3-overhangs in a length-dependent manner, with its impact increasing for longer reverse-transcribed overhangs. This understanding reveals that regulating Artemis activity can improve insertion frequency specifically for PRINS. We develop and validate robust epigenetic (CRISPRoff) and antisense oligonucleotide (ASO) strategies to effectively silence/knockdown Artemis expression, successfully recapitulating the beneficial PRINS editing outcomes observed in Artemis-deficient cells. Leveraging these insights, we show that Artemis modulation can enhance endogenous protein tagging in cells, including challenging hiPSC-derived non-dividing cardiomyocytes. Our findings support Artemis as a key regulator of PRINS editing outcomes and present a tunable strategy to optimize insertion efficiency for diverse genomic engineering and therapeutic applications.

molecular biology↗

bioRxiv2024

Transgenic augmentation of erythroferrone in mice ameliorates anemia in adenine-induced chronic kidney disease

Anemia is a common and disabling complication of chronic kidney disease (CKD). Current therapies can be burdensome, and full correction of anemia is limited by cardiovascular side effects. New approaches that may offer additional therapeutic options are needed. We explored the anti-anemic effects of erythroferrone, an erythroid hormone that induces iron mobilization by suppressing the master iron-regulatory hormone hepcidin. In a preclinical murine model of adenine-induced CKD, transgenic augmentation of erythroferrone mobilized iron, increased hemoglobin concentrations by approximately 2 g/dl, and modestly improved renal function without affecting systemic or renal inflammation, fibrosis, or markers of mineral metabolism. This study supports the concept that therapeutic augmentation of erythroferrone is a promising approach for alleviating CKD-associated anemia.

pathology↗

bioRxiv2023

Multifaceted proteome analysis at solubility, redox, and expression dimensions for target identification

Multifaceted interrogation of the proteome deepens the system-wide understanding of biological systems; however, mapping the redox changes in the proteome has so far been significantly more challenging than expression and solubility/stability analyses. Here, we devise the first high- throughput redox proteomics approach integrated with expression analysis (REX) and combine it with Proteome Integral Solubility Alteration (PISA) assay. The whole PISA-REX experiment with up to four biological replicates can be multiplexed into a single tandem mass tag TMTpro set. For benchmarking this compact tool, we analyzed HCT116 cells treated with auranofin, showing great improvement compared with previous such studies. Then we applied PISA-REX to study proteome remodeling upon stimulation of human monocytes by interferon . We also studied the proteome changes in plasmacytoid dendritic cells isolated from wild type vs. Ncf1- mutant mice treated with interferon , showing that NCF1 deficiency enhances the STAT1 pathway and modulates the expression, solubility and redox state of interferon-induced proteins. Providing comprehensive multifaceted information on the proteome, the compact PISA-REX has the potential to become an industry standard in proteomics and to open new windows into the biology of health and disease.

biochemistry↗

bioRxiv2022

Simultaneous inhibition of DNA-PK and Pol{Theta} improves integration efficiency and precision of genome editing

Genome editing tools, especially CRISPR/Cas9-based strategies, have transformed biomedical research and opened opportunities for developing curative treatments for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of undesired mutations represent major limitations for genome editing applications. Both issues arise from the interplay between the main DNA Double-Strand Break (DSB) repair pathways, Homology-Directed Repair (HDR), Non-Homologous End Joining (NHEJ), and Microhomology-Mediated End Joining (MMEJ). To improve efficiencies of targeted CRISPR-Cas9 genome editing, we screened a large compound library. This led to the discovery of AZD7648, a DNA-dependent protein kinase (DNA-PK) inhibitor and potent enhancer of CRISPR-Cas9-mediated integration. We demonstrated that AZD7648 increased HDR and decreased mutagenic NHEJ repair, thus resulting in improved performance of precise gene editing. Furthermore, we observed additional improvement of integration efficiency by impairing MMEJ repair through DNA polymerase {ominus} (Pol{ominus}) inhibition. Combined treatment with AZD7648 and Pol{ominus} inhibitors (which we named 2iHDR) substantially increased precision of templated insertions, with efficiencies of up to 80%, and nearly no formation of undesired Insertion-Deletions (InDels). Importantly, 2iHDR also decreased Cas9-associated off-target activity, dramatically improving the performance and fidelity of CRISPR-Cas9 gene editing.

molecular biology↗

bioRxiv2020

ObLiGaRe doxycycline Inducible (ODIn) Cas9 system driving pre-clinical drug discovery, from design to cancer treatment

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. We have generated a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene. Targeted ObLiGaRe resulted in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse can be utilized for robust and tunable genome editing allowing for flexibility, speed and uniformity at reduced cost, leading to high throughput and practical preclinical in vivo therapeutic testing.

genomics↗