Transposition is normally considered a DNA-based process. Now, scientists have devised a means to carry out a similar transformation — this time using proteins. The approach enables protein semisynthesis and site-specific modification of folded substrates in vitro and in live cells, opening up new ways to probe and manipulate biological systems.

Nano-carriers are revolutionizing plant genetic engineering by facilitating the direct and efficient delivery of nucleic acids and proteins into plant cells. By precisely controlling and tailoring their properties, these carriers can effectively penetrate the plant cell wall, delivering bioactive molecules overcoming the constraints imposed by plant genotype specificity. This advancement provides a powerful, flexible, and broadly applicable strategy for accelerating crop health and resilience. Conventional plant transformation methods, such as Agrobacterium-mediated gene transfer and biolistic delivery, are limited by species-specific constraints, lengthy regeneration processes, and increased risks of genomic instability. Nanomaterial-based delivery systems offer a flexible alternative but face the significant challenge of penetrating the complex, multilayered plant cell wall. Recent research highlights three effective strategies: optimizing particle size, adjusting surface charge, and using cell wall-degrading enzymes. Together, these approaches greatly enhance nanocarrier uptake, enabling delivery of diverse bioactive agents like nucleic acids, proteins and gene-editing tools making nanotechnology a promising tool for crop improvement.

The gut virome plays a pivotal role in shaping the host’s microbiota. However, gut viruses across different mammalian models, and their connections with the human gut microbiota remain largely unknown. We identified 977 high-confidence species-level viral operational taxonomic units (vOTUs) in mice (hcMGV), 12,896 in pigs (hcPGV), and 1480 in cynomolgus macaques (hcCMGV) from metagenomes, respectively. Clustering these vOTUs at approximately genus level uncovered novel clades with high prevalence across animal guts (> = 60%). In particular, crAss-like phages and cas-harboring jumbophages were characterized. Comparative analysis revealed that hcCMGV had a closer relationship with hcPGV than hcMGV, despite the animal-specific characteristics, and that 55.88% hcCMGV had connections with the human microbiota. Our findings shed light on the diversity of gut viruses across these three animals, contributing to future gut microbial studies using model animals.

CRISPR-Cas diagnostics are revolutionizing point-of-care molecular testing due to the programmability, simplicity, and sensitivity of Cas systems with trans-cleavage activity. CRISPR-Cas12 assays are promising for detecting single nucleotide polymorphisms (SNPs). However, reports vary widely describing Cas12 SNP sensitivity, and an underlying mechanism is lacking. We systematically varied crRNA length and valency to investigate the role of crRNA architectures on Cas12 biosensing in the context of speed-of-detection, sensitivity, and selectivity. Our results demonstrate that crRNAs complementary to 20 base pairs of the target DNA is optimal for rapid and sensitive detection, while a complementary length of 15 base pairs is ideal for robust SNP detection. Additionally, we uncovered a unique periodicity in SNP sensitivity based on nucleotide position and developed a structural model explaining what drives Cas12 SNP sensitivity. Lastly, we showed that bivalent CRISPR-Cas sensors have synergistic and enhanced activity that is distance dependent. Profiling crRNAs revealed optimal length for Cas12-based molecular testing and SNP detection. Investigation into multivalent architectures elucidated valency and distance dependence for synergistic Cas12 activity.

Traditionally perceived as an RNA-specific nuclease, Cas13a has been used primarily for RNA detection. We discover the ability of Leptotrichia buccalis Cas13a (LbuCas13a) to directly target DNA without the restrictions of protospacer flanking sequence and PAM sequences, coupled with robust trans-cleavage activity. Contrary to conventional understanding, LbuCas13a does not degrade DNA targets. Our study reveals an enhancement in the single-nucleotide specificity of LbuCas13a against DNA compared to RNA. This heightened specificity is attributed to the lower affinity of CRISPR RNA (crRNA) towards DNA, raising the crRNA–DNA binding energy barrier. We introduce a molecular diagnostic platform called superior universal rapid enhanced specificity test with LbuCas13a (SUREST) for high-resolution genotyping. SUREST is capable of detecting DNA concentrations of CYP2C19 (rs4986893) as minute as 0.3 aM (0.18 cps µl−1). We also apply SUREST to human genotyping scenarios, indicating that SUREST performs well across a broad range of mutations and sequence contexts. SUREST represents an advancement in real-time nucleic acid detection, making it a useful tool for pathogen identification and mutation analysis in clinical diagnostics. A Leptotrichia buccalis Cas13a diagnostic platform can be used to cleave and detect small concentrations of viral and human DNA.

RNA-driven phase separation is emerging as a promising approach for engineering biomolecular condensates with diverse functionalities. Condensates form thanks to weak yet specific RNA–RNA interactions established by design via complementary sequence domains. Here, we demonstrate how RNA condensates formed by star-shaped RNA motifs, or nanostars, can be dynamically controlled when the motifs include additional linear or branch-loop domains that facilitate access of regulatory RNA molecules to the nanostar interaction domains. We show that condensates dissolve in the presence of RNA “invaders” that occlude selected nanostar bonds and reduce the valency of the nanostars, preventing phase separation. We further demonstrate that the introduction of “anti-invader” strands, complementary to the invaders, makes it possible to restore condensate formation. An important aspect of our experiments is that we demonstrate these behaviors in one-pot reactions, where RNA nanostars, invaders, and anti-invaders are simultaneously transcribed in vitro using short DNA templates. Our results lay the groundwork for engineering RNA-based assemblies with tunable, reversible condensation, providing a promising toolkit for synthetic biology applications requiring responsive, self-organizing biomolecular materials.

Glutamate dehydrogenase (GDH) resides at the crossroads of nitrogen and carbon metabolism, catalyzing the reversible conversion of L-glutamate to α-ketoglutarate and ammonium. GDH paralogs are ubiquitous across most species, presumably enabling functional specialization and genetic compensation in response to diverse conditions. Staphylococcus aureus harbors a single housekeeping GDH (GudB), whereas Bacillus subtilis encodes both a major and a minor GDH, GudB and RocG, respectively. In an unsuccessful attempt to identify an alternative GDH in S. aureus, we serendipitously discovered previously unrecognized GDH activity in two metabolic enzymes of B. subtilis. The hexameric Val/Leu/Ile dehydrogenase Bcd (formerly YqiT) catabolizes branched-chain amino acids and to a lesser extent glutamate using NAD+ as a cofactor. Removal of gudB and rocG unmasks the dual NAD(P)+-dependent GDH activity of RocA, which otherwise functions as a 3-hydroxy-1-pyrroline-5-carboxylate dehydrogenase. Bcd homologs are prevalent in free-living and obligate bacteria but are absent in most, if not all, staphylococci. Despite low sequence homology, Bcd structurally resembles the GudB/RocG family and can functionally compensate for the loss of GudB in S. aureus. Bcd is essential for the full maturation of biofilms. B. subtilis lacking GDHs exhibits severe impairments in rugose architecture and colony expansion of biofilms. This study underscores the importance of metabolic redundancy and highlights the critical role of substrate promiscuity in GDHs during biofilm development.