Parikh, S. J. | bioRxiv Science

bioRxiv2025

Targeted control of gene expression using CRISPR-associated endoribonucleases

CRISPR-associated endoribonucleases (Cas RNases) cleave single-stranded RNA in a highly sequence-specific manner, by recognizing and binding to short RNA sequences known as direct repeats (DRs). Here we investigate the potential of exploiting Cas RNases for the regulation of target genes with one or more DRs introduced into the 3 untranslated region, an approach we refer to as DREDGE (direct repeat-enabled down-regulation of gene expression). The DNase-dead version of Cas12a (dCas12a) was identified as the most efficient among 5 different Cas RNases tested and was subsequently evaluated in doxycycline-regulatable systems targeting either stably expressed fluorescent proteins or an endogenous gene. DREDGE performed superbly in stable cell lines, resulting in up to 90% downregulation with rapid onset, notably, in a fully reversible manner. Successful control of an endogenous gene with DREDGE was demonstrated in two formats, including one wherein both the DR and the transgene driving expression of dCas12a were introduced in one step by CRISPR-Cas. Our results establish DREDGE as an effective method for regulating gene expression in a targeted, highly selective, and fully reversible manner, with several advantages over existing technologies.

molecular biology↗

bioRxiv2020

Viromes outperform total metagenomes in revealing the spatiotemporal patterns of agricultural soil viral communities

Viruses are abundant yet understudied members of soil environments that influence terrestrial biogeochemical cycles. Here, we characterized the dsDNA viral diversity in biochar-amended agricultural soils at the pre-planting and harvesting stages of a tomato growing season via paired total metagenomes and viromes. Size fractionation prior to DNA extraction reduced sources of non-viral DNA in viromes, enabling the recovery of a vaster richness of viral populations (vOTUs), greater viral taxonomic diversity, broader range of predicted hosts, and better access to the rare virosphere, relative to total metagenomes, which tended to recover only the most persistent and abundant vOTUs. Of 2,961 detected vOTUs, 2,684 were recovered exclusively from viromes, while only three were recovered from total metagenomes alone. Both viral and microbial communities differed significantly over time, suggesting a coupled response to rhizosphere recruitment processes and nitrogen amendments. Viral communities alone were also structured along a spatial gradient. Overall, our results highlight the utility of soil viromics and reveal similarities between viral and microbial community dynamics throughout the tomato growing season yet suggest a partial decoupling of the processes driving their spatial distributions, potentially due to differences in dispersal, decay rates, and/or sensitivities to soil heterogeneity.

microbiology↗