Biology subjects

Nysten, J.

Publications and source records attributed to Nysten, J..

3 recordsLinked to original sources

bioRxiv2026

Systematic dissection of Cas12a-mediated precision genome editing defines design principles for genome-scale variant engineering

Cas9 precision editing is increasingly predictable because guide, donor and target-context effects have been systematically characterized. Extending this framework to other nucleases is essential for installing variants outside convenient Cas9 target space. Cas12a provides a T-rich protospacer-adjacent motif (PAM) alternative, but determinants of efficient donor-templated Cas12a editing remain poorly defined. Here, we systematically dissected Cas12a precision editing in Saccharomyces cerevisiae across nuclease, direct repeat, expression, crRNA, donor, genomic context and time-course variables. Reporter and amplicon-sequencing assays showed that cleavage activity alone did not predict precise editing. Highly active configurations often reduced viability or lost edited alleles over time, whereas attenuated configurations better preserved programmed edits. Enhanced AsCas12a edited rapidly and tolerated shorter crRNAs, resulting in a narrower editing window, while an attenuated FnCas12a configuration edited more slowly but maintained higher viability and better distal-edit recovery. Alternative repair outcomes were rare, target-dependent, and further suppressed by LexA-FHA donor recruitment. To define design parameters at scale, we established a pooled Cas12a platform with 530 barcoded edit cassettes and recovered programmed edits for 70.2% of designs. Successful editing was reduced with TTTG PAMs, a C upstream of the PAM and at distal edit positions. Excluding these features increased the edited fraction to 85.4% and adding high predicted cleavage scores further elevated it to 91.4%. Applied retrospectively, these criteria also identified poorly edited loci in the targeted panels. Together, these data define design principles for Cas12a-mediated precision editing and establish a scalable platform for genome-scale pooled variant engineering and phenotyping in yeast.

Synthetic Biology↗

bioRxiv2025

Expansion, functional diversification and gene fusion events in the Ato protein family

Candida albicans, a commensal opportunistic pathogen, exhibits remarkable metabolic flexibility and adaptability to environmental changes. In glucose-limited niches, it utilizes alternative carbon sources such as carboxylic acids, which may influence its pathogenicity. In Saccharomyces cerevisiae, the uptake of monocarboxylates occurs through regulated plasma membrane (PM) transport proteins, such as Ato1 (Ady2), which belongs to the Acetate Uptake Transporter (AceTr) family. In C. albicans, these proteins are notably expanded, consisting of ten Ato-like proteins (ATO1-ATO10), whose functions remain unknown. Here, we investigated the role of Ato proteins in carboxylic acid utilization by C. albicans using in-silico and functional analysis. Our data revealed that several C. albicans Atos retain conserved AceTr motifs but possess distinct structural features, including differences in pore radius and binding sites for acetate and lactate. Expression analysis revealed that Ato1, Ato2, Ato3, and Ato6 exhibit distinct cellular localization and expression levels on the plasma membrane, depending on the presence or absence of monocarboxylates. Remarkably, deletion of ATO1 impaired Ato2 and Ato3 expression and caused ER retention of a distinct form of Ato2, suggesting a central regulatory role for Ato1 in the Ato transport system. Finally, we identified a novel Ato-related protein family in vertebrates. This family has three consecutive 6-helix transport domains and a unique C-terminal fusion with Sua5/YciO/YrdC, an enzyme involved in tRNA modification. Overall, our data suggests that the Ato protein family might play a critical role in the utilization of acetic or lactic acids in C. albicans. It also proposes potential functional redundancy among its members, which may contribute to rapid environmental adaptation and pathogenicity.

microbiology↗

bioRxiv2024

The riboflavin biosynthetic pathway as a novel target for antifungal drugs against Candida species

In recent decades, there has been an increase in the occurrence of fungal infections, yet the arsenal of drugs available to fight invasive infections remains very limited. The development of new antifungal agents is hindered by the restricted number of molecular targets that can be exploited, given the shared eukaryotic nature of fungi and their hosts which often leads to host toxicity. In this paper, we examine the riboflavin biosynthetic pathway as a potential novel drug target. Riboflavin is an essential nutrient for all living organisms. Its biosynthetic pathway does not exist in humans, who obtain riboflavin through their diet. Our findings demonstrate that all enzymes in the pathway are essential for Candida albicans, Candida glabrata, and Saccharomyces cerevisiae. Among these enzymes, Rib1 and Rib3 are the most promising targets. Auxotrophic strains, which mimic a drug targeting the biosynthesis pathway, experience rapid mortality in the absence of supplemented riboflavin. Nevertheless, the cells can still take up external riboflavin when supplemented. We identified Orf19.4337 as the riboflavin importer in C. albicans and named it Rut1. We found that Rut1 only facilitates growth at external riboflavin concentrations that exceed the physiological concentrations in the human body, making it unlikely that riboflavin uptake to act as a potential resistance mechanism for a drug targeting the biosynthesis pathway. Interestingly, the uptake system in S. cerevisiae is more effective than in C. albicans and C. glabrata, enabling an auxotrophic S. cerevisiae strain to outcompete an auxotrophic C. albicans strain in lower riboflavin concentrations. ImportanceCandida species are a common cause of invasive fungal infections. Candida albicans, in particular, poses a significant threat to immunocompromised individuals. This opportunistic pathogen typically lives as a commensal on mucosal surfaces of healthy individuals, but it can also cause invasive infections associated with high morbidity and mortality. Currently, there are only three major classes of antifungal drugs available to treat these infections. Additionally, the efficacy of these antifungal agents is restricted by host toxicity, suboptimal pharmacokinetics, a narrow spectrum of activity, intrinsic resistance of fungal species, such as Candida glabrata, to certain drugs, and the acquisition of resistance over time. Therefore, it is crucial to identify new antifungal drug targets with novel modes of action to add to the limited armamentarium.

microbiology↗