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Biology subjects

Peterka, M.

Publications and source records attributed to Peterka, M..

10 recordsLinked to original sources
bioRxiv2026

Enhanced γ-globin reactivation and sickle cell correction through a repressor-to- activator motif switch in the HBG1/2 promoters

Sickle cell disease (SCD) is caused by the production of an abnormal adult hemoglobin that generates sickle-shaped red blood cells (RBCs). Transplantation of autologous genetically corrected hematopoietic stem/progenitor cells (HSPCs) represents a promising therapy. Persistent fetal hemoglobin expression improves SCD. Here, we engineered the fetal HBG1/2 promoters by replacing the BCL11A repressor binding site (BS) with a TAL1:GATA1 motif recognized by transcriptional activators. We exploited the prime editing nuclease (PEn) that efficiently installed the TAL1:GATA1 motif in K562 cells, outperforming the original PE. Non-homologous end joining (NHEJ) and/or alternative-end joining (alt-EJ) pathway inhibition enhanced precise editing. However, this strategy was poorly efficient in patients HSPCs. Alternatively, we used CRISPR/Cas9 nuclease to either disrupt the BCL11A BS via NHEJ and/or alt-EJ or to replace it with the TAL1:GATA1 motif via homology-directed repair (HDR) using a donor ssODN template. NHEJ and alt-EJ inhibition improved product purity, reducing InDels and achieving superior precise editing efficiency compared to PEn in K562 and HSPCs. HDR-edited HSPCs preserved clonogenic capacity and differentiated into RBCs showing elevated HBG expression and correction of the sickling phenotype. These results demonstrate that replacing the BCL11A BS with a TAL1:GATA1 motif is a potent strategy for reactivating HBG1/2 to treat SCD.

bioengineering↗
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↗
bioRxiv2025

Purification and Immunogenicity of Nipah Virus-Like Particles from Insect Cells

Nipah virus (NiV) is an emerging high-fatality zoonotic threat lacking approved vaccines. Current virus-like particle (VLP) production methods rely on costly mammalian cell systems and non-scalable ultracentrifugation purification. We developed an efficient production system for enveloped NiVLPs by co-expressing structural proteins F, G, and M using a baculovirus expression vector system in Sf9 cells. A novel multi-step chromatographic purification process was established using monolith convective media, integrating steric exclusion chromatography with sequential cation and anion exchange steps. Purified NiVLPs were characterized by nanoparticle tracking analysis and transmission electron microscopy, then evaluated for immunogenicity in Syrian golden hamsters. The optimized process yielded enveloped particles of approximately 100-120 nm that morphologically resemble native NiV virions. A single 25 g NiVLP dose induced robust systemic anti-NiV G IgG responses within 14 days, demonstrating rapid immunogenicity suitable for outbreak response. However, neutralizing antibody titers against NiV remained limited compared to total IgG responses. This study establishes the first chromatography-based manufacturing platform for morphologically correct NiVLPs from insect cells. A deeper understanding of the immunity generated is needed to support their potential as a rapidly deployable vaccine platform against NiV.

bioengineering↗
bioRxiv2025

Optimised genome editing for precise DNA insertion and substitution using Prime Editors in zebrafish

CRISPR/Cas9-mediated genome editing has rapidly become a popular tool for studying gene functions and generating genetically modified organisms. However, using this system, stochastic integration of random insertions and deletions restricts precise genome manipulation. Advanced CRISPR/Cas9 technologies using Prime Editors (PEs), Cas9 proteins fused with reverse transcriptase, enable programmed integration of short DNA modifications into the genome. However, its application in precise genome editing in animal models is challenging. Here, we utilise a nickase-and a nuclease-based PE to perform programmed short DNA substitution and insertion in various loci in the zebrafish genome. Whereas nickase-based PE2 mediated a higher ratio of precise prime edits to the total edits, nuclease-based PEn was more efficient for short DNA modifications, achieving up to 27.3% precise insertion. To further evaluate our approach, we inserted a nuclear localisation signal into a reporter transgene to incorporate longer fragments by prime editing. These gene modifications were transmitted to the next generation. We show that PE-mediated prime editing can efficiently manipulate genome information in zebrafish without using exogenous donor DNA. Prime editing, CRISPR/Cas9, zebrafish, PE2, PEn

molecular biology↗
bioRxiv2024

Improved nuclease-based prime editing by DNA repair modulation and pegRNA engineering

Prime editing is a genome engineering tool that allows installation of small edits with high precision. However, prime editing efficiency and purity can vary widely across different edits, genomic targets, and cell types. Prime editing nuclease (PEn) utilizes a fully active Cas9 instead of the nickase employed in conventional prime editors. PEn is capable of editing sites resistant to nickase-based prime editors but induces more undesired editing events. In this work, we introduce two strategies to enhance PEn precision and efficiency. First, we apply a small molecule approach, selectively modulating DNA repair pathways, to improve PEn precision up to 9.8-fold and reduce off-target editing by 90%. Second, through pegRNA engineering, we devise a strategy that mitigates unintended pegRNA scaffold integration, which is a common prime editing by-product, enhancing precision up to 3.5-fold. We apply this approach to a specific type of PEn editing mediated through non-homologous end joining and use it to achieve efficient and precise prime editing in multiple human cell lines, primary human hepatocytes, and mouse embryos. Together, this work presents two general strategies to improve prime editing, overcomes the limitations of current PEn editors, and provides reliable and precise genome editing outcomes, a pivotal requirement for therapeutic applications.

bioengineering↗
bioRxiv2023

Shared Structural Features of Miro Binding Control Mitochondrial Homeostasis

Miro proteins are universally conserved mitochondrial calcium-binding GTPases that regulate a multitude of mitochondrial processes, including transport, clearance and lipid trafficking. Miro binds a variety of client proteins involved in these functions. How this binding is operated at the molecular level and whether and how it is important for mitochondrial health, however, remains unknown. Here, we show that known Miro clients all use a similar short motif to bind the same structural element: a highly conserved hydrophobic pocket in the calcium-binding domain of Miro. Using these Miro-binding motifs, we identified direct interactors de novo, including yeast Mdm34, and mammalian MTFR1/2/1L, VPS13D and Parkin. Given the shared binding mechanism and conservation across eukaryotes, we propose that Miro is a universal mitochondrial adaptor coordinating mitochondrial health. One-Sentence SummaryFunctionally diverse mitochondrial proteins interact with a conserved hydrophobic pocket on the calcium-binding Miro-GTPases.

biochemistry↗
bioRxiv2021

Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing

Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two novel strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing lead to increased levels of by-products, it rescued pegRNAs that performed poorly with a nickase-based prime editor. We also present a small molecule approach that yielded increased product purity of PEn editing. Next, we developed a homology-independent PEn editing strategy by engineering a single primed insertion gRNA (springRNA) which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.

molecular biology↗
bioRxiv2018

Miro-dependent mitochondrial pool of CENP-F and its farnesylated C-terminal domain are dispensable for normal development in mice

CENP-F is a large, microtubule-binding protein that regulates multiple cellular processes including chromosome segregation and mitochondrial trafficking at cytokinesis. This multiplicity of function is mediated through the binding of various partners, like Bub1 at the kinetochore and Miro at mito-chondria. Due to the multifunctionality of CENP-F, the cellular phenotypes observed upon its depletion are difficult to interpret and there is a need to genetically separate its different functions by preventing binding to selected partners. Here we engineer a CENP-F point-mutant that is deficient in Miro binding and thus is unable to localize to mitochondria, but retains other localizations. We introduced this mutation in cultured human cells using CRISPR/Cas9 and show it causes a defect in mitochondrial spreading similar to that observed upon Miro depletion. We further create a mouse model carrying this CENP-F variant, as well as truncated CENP-F mutants lacking the farnesylated C-terminus of the protein. Importantly, one of these truncations leads to [~]80% downregulation of CENP-F expression. We observe that, despite the phenotypes apparent in cultured cells, mutant mice develop normally. Taken together, these mice will serve as important models to study CENP-F biology at organismal level. In addition, because truncations of CENP-F in humans cause a lethal disease termed Stromme syndrome and because CENP-F is involved in cancer development, they might also be relevant disease models.

cell biology↗
bioRxiv2016

CENP-F couples cargo to growing and shortening microtubule ends

Dynamic microtubule ends exert pulling and pushing forces on intracellular membranes and organelles. However, the mechanical linkage of microtubule tips to their cargoes is poorly understood. CENP-F is a non-motor microtubule-binding protein that participates in microtubule binding at kinetochores and in the mitotic redistribution of the mitochondrial network. CENP-F-driven mitochondrial transport is linked to growing microtubule tips, but the underlying molecular mechanisms are unknown. Here we show that CENP-F tracks growing microtubule ends in living cells. In vitro reconstitution demonstrates that microtubule tips can transport mitochondria and CENP-F-coated artificial cargoes over micrometer-long distances, during both growing and shrinking phases. Based on these and previous observations, we suggest that CENP-F might act as a transporter of mitochondria and other cellular cargoes by attaching them to dynamic microtubule ends.

cell biology↗