Biology subjects

Kutnowski, N.

Publications and source records attributed to Kutnowski, N..

3 recordsLinked to original sources

bioRxiv2025

StabilizeIT: An Automated Workflow for Protein Stabilization

The industrial application of enzymes is often hampered by poor stability and low expression yields. While computational tools can predict stabilizing mutations, many are bound by restrictive licenses that hinder their broader adoption. To address this, we developed StabilizeIT, a powerful, open-access webserver for enhancing protein stability and expression. StabilizeIT integrates a pipeline of curated open-source tools such as ProteinMPNN, AlphaFold2 and SaProt with our state-of-the-art model, SolvIT, which accurately predicts heterologous expression titers in E. coli. This unique combination allows for the simultaneous optimization of melting temperature (Tm) and solubility. The pipeline exhibits remarkable speed, generating dozens of high-quality candidates with predicted high titers and increased stability in under an hour, streamlining the path to experimental validation. To demonstrate its efficacy, StabilizeIT was used to engineer multiple enzymes in our novel biosynthetic pathway for Hyaluronic Acid. The resulting variants showed greatly enhanced thermal stability and expression, proving the pipelines real-world utility. StabilizeIT is now available to the community, offering an accessible and validated solution to accelerate the development of robust proteins for diverse applications. The webserver is freely available at https://stabilizeit.enzymit.com

bioinformatics↗

bioRxiv2024

Activity of zebrafish THAP9 transposase and zebrafish P element-like transposons

Transposable elements are mobile DNA segments that are found ubiquitously across the three domains of life. One family of transposons, called P elements, were discovered in the fruit fly Drosophila melanogaster. Since their discovery, P element transposase-homologous genes (called THAP-domain containing 9 or THAP9) have been discovered in other animal genomes. Here, we show that the zebrafish (Danio rerio) genome contains both an active THAP9 transposase (zfTHAP9) and mobile P-like transposable elements (called Pdre). zfTHAP9 transposase can excise one of its own elements (Pdre2) and Drosophila P elements. Drosophila P element transposase (DmTNP) is also able to excise the zebrafish Pdre2 element, even though its distinct from the Drosophila P element. However, zfTHAP9 cannot transpose Pdre2 or Drosophila P elements, indicating partial transposase activity. Characterization of the N-terminal THAP DNA binding domain of zfTHAP9 shows distinct DNA binding site preferences from DmTNP and mutation of the zfTHAP9, based on known mutations in DmTNP, generated a hyperactive protein. These results define an active vertebrate THAP9 transposase that can act on the endogenous zebrafish Pdre and Drosophila P elements.

biochemistry↗

bioRxiv2022

Highly efficient generation of isogenic pluripotent stem cell models using prime editing

The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multi-component editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair (HDR) of site-specific nuclease-induced double-strand breaks (DSBs). Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated chemically-modified pegRNAs and nicking guide RNAs (ngRNAs) improved editing efficiencies up to 13-fold compared to transfecting the prime editing components as plasmids or ribonucleoprotein particles (RNPs). Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinsons disease in hPSCs.

molecular biology↗