Biology subjects

Montagud-Martinez, R.

Publications and source records attributed to Montagud-Martinez, R..

3 recordsLinked to original sources

bioRxiv2026

Amplification-free CRISPR/Cas13a-based viroid detection in RNA extracts from infected plants

Viroids are plant infectious agents that threaten agricultural production. Current viroid detection methods rely on RT-PCR-based assays, which require specialized laboratory equipment and can sometimes produce false-negative results or non-specific amplification due to the high sequence conservation among closely related viroid species. CRISPR-based diagnostics, particularly Cas12-based systems for DNA detection (DETECTR) and Cas13a-based systems (SHERLOCK) for RNA detection, have emerged as powerful tools for nucleic acid diagnostics. However, most existing workflows still rely on target amplification and, in the case of Cas13a systems, require additional in vitro transcription steps, limiting their simplicity and direct applicability for plant diagnostics. Here, we developed a direct amplification-free Cas13a-based detection platform for viroids using potato spindle tuber viroid (PSTVd) as a model. We optimized CRISPR RNA (crRNA) design, identified inhibitory effects of plant total RNA on readout signal, and employed simplified viroid RNA enrichment workflows enabling robust detection in plant samples. The system further supported both PSTVd-specific and broad-spectrum pospiviroid (genus Pospiviroid) detection and was successfully extended to avocado sunblotch viroid (family Avsunviroidae), demonstrating its adaptability across distinct viroid families. Together, these results establish a practical and modular Cas13a-based platform, not only for viroid diagnostics, but also for broader applications in RNA-derived plant pathogen detection. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/736049v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1b62802org.highwire.dtl.DTLVardef@1fcd8b4org.highwire.dtl.DTLVardef@f73e8dorg.highwire.dtl.DTLVardef@2ba7c9_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance statementA simplified RNA enrichment workflow combined with CRISPR-Cas13a enables direct, amplification-free detection of plant viroids. The assay supports early and reliable diagnosis across different tomato varieties and provides a practical strategy for improving molecular detection of plant pathogens.

plant biology↗

bioRxiv2025

CRISPR-Cas9 trans-cleavage is hindered by a closed R-loop, an elongated spacer, and an inactive HNH domain

Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key structural determinants underlying this function. First, we show that open R-loops in the PAM-distal region favor trans-cleavage activity, which occur when targeting short double-stranded or single-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impairs this collateral activity. Third, although trans-cleavage is mediated by the RuvC domain, we show that a catalytically active HNH domain contributes to an efficient process. Structural analyses of domain rearrangements provide mechanistic insight. Together, these findings illustrate a fine modulation of Cas9 function.

biochemistry↗

bioRxiv2022

Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria

The RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expression in the bacterium Escherichia coli with the mammalian RNA-binding protein Musashi-1, which is a stem cell marker with neurodevelopmental role that contains two canonical RRMs. In the circuit, Musashi-1 is regulated transcriptionally and works as an allosteric translation repressor thanks to a specific interaction with the N-terminal coding region of a messenger RNA and its structural plasticity to respond to fatty acids. We fully characterized the genetic system at the population and single-cell levels showing a significant fold change in reporter expression, and the underlying molecular mechanism by assessing the in vitro binding kinetics and in vivo functionality of a series of RNA mutants. The dynamic response of the system was well recapitulated by a bottom-up mathematical model. Moreover, we applied the post-transcriptional mechanism engineered with Musashi-1 to specifically regulate a gene within an operon, to implement combinatorial regulation, and to reduce protein expression noise. This work illustrates how RRM-based regulation can be adapted to simple organisms, thereby adding a new regulatory layer in prokaryotes for translation control.

synthetic biology↗