Biology subjects

Nasrollahi, V.

Publications and source records attributed to Nasrollahi, V..

3 recordsLinked to original sources

bioRxiv2025

Conjugation Based CRISPR Antifungals (COBRA)

Fungal infections are increasingly difficult to treat due to rising antifungal resistance and the limited number of effective drug classes. To address this challenge, we developed a conjugation-based CRISPR antifungal (COBRA) platform that enables delivery of programmable gene-targeting machinery from Escherichia coli to Saccharomyces cerevisiae. We engineered a mobilizable pVenom plasmids containing an oriT for conjugation, Cas9 and guide RNAs for elimination of yeast. To prevent toxicity in E. coli, yeast ACT1 intron is inserted in Cas9. Seven guide RNAs targeting essential genes involved in cell cycle progression, ribosome function, and DNA replication were first assessed by using electroporation as delivery and demonstrated strong lethality for guides targeting CDC28 and MCM2. When delivered by conjugation, these guides again reproduced these results, whereas an intron-targeting control remained non-lethal. Dual-guide plasmids eliminated yeast colony formation entirely. Together, these findings demonstrate that conjugation-delivered CRISPR machinery can successfully target endogenous fungal genes and, especially when multiplexed, offers an effective and programmable antifungal strategy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/692781v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1fce050org.highwire.dtl.DTLVardef@ead257org.highwire.dtl.DTLVardef@978391org.highwire.dtl.DTLVardef@13ead19_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Conjugation-mediated DNA delivery to the filamentous fungus Ustilago maydis

Phytopathogenic fungi are ubiquitous throughout the environment and threaten global food security. This issue is further amplified by the increasing resistance of pathogens to antimicrobials. Current chemical-based antifungals target cells by inhibiting growth or metabolic function, making them ideal for fungal gain of resistance mutations. Biofungicides are a rising class of antifungals that have low potential for negative environmental impact and provide the fungi almost no potential for gaining resistance. Conjugative plasmids which play a role in the natural mechanism of horizontal gene transfer in bacteria, have been repurposed to deliver toxic genetic cargo to recipient cells, showing promise as next-generation antimicrobial agents. In this work, we have demonstrated the first protocol for delivering DNA from Escherichia coli to the filamentous phytopathogen, Ustilago maydis through conjugation. DNA delivery was confirmed using PCR screening of DNA isolated from the re-streaked transconjugants. Although challenges such as reduced conjugation efficiency and extrachromosomal replication persist, this work establishes the first step towards creating a conjugation-based biofungicide.

synthetic biology↗

bioRxiv2025

Engineering Conjugative Plasmids for Inducible Horizontal DNA Transfer

Rapidly developing microbial resistance to existing antimicrobials poses a growing threat to public health and global food security. Current chemical-based treatments target cells by inhibiting growth or metabolic function, but their effectiveness is diminishing. To address the growing antimicrobial resistance crisis, there is an urgent need for innovative therapies. Conjugative plasmids, a natural mechanism of horizontal gene transfer in bacteria, have been repurposed to deliver toxic genetic cargo to recipient cells, showing promise as next-generation antimicrobial agents. However, the ecological risks posed by unintended gene transfer require robust biocontainment strategies. In this study, we developed inducible conjugative plasmids to solve these challenges. Utilizing an arabinose-inducible promoter, we evaluated 13 plasmids with single essential gene deletions, identifying trbC and trbF as strong candidates for stringent regulation. These plasmids demonstrated inducibility in both cis and trans configurations, with induction resulting in up to a 5-log increase in conjugation efficiency compared to uninduced conditions. Although challenges such as reduced conjugation efficiency and promoter leakiness persist, this work establishes a foundation for the controlled transfer of plasmids, paving the way for safer and more effective antimicrobial technologies.

synthetic biology↗