Snake venoms are complex mixtures of toxic proteins with important medical and pharmacological relevance. To explore the genetic diversity of these venoms, VenomCap was created—an exon-capture probe set targeting toxin-coding genes in various elapid snakes, especially the Hydrophiinae subfamily. Testing on 24 species showed VenomCap’s success in recovering exons from about 1500 target markers, covering 24 known venom gene families. The results were particularly robust among hydrophiines, demonstrating its effectiveness as a cost-efficient alternative to full genome sequencing. VenomCap facilitates the study of venom gene diversity and evolution, with potential applications across other venomous snake families.

Phylogenomic data are enhancing insect phylogenetics, particularly through target enrichment methods. This study introduces an Orthoptera-specific target enrichment (OR-TE) probe set developed from 80 transcriptomes, targeting 1828 loci with diverse evolutionary rates. The probe set was validated by generating phylogenomic data from 36 previously unstudied orthopteran species, capturing an average of 1037 loci and clarifying phylogenetic relationships. Documentation of the probe design and bioinformatics process aims to promote broader adoption of this tool.

Antarctic krill (Euphausia superba) is a crucial species in the Southern Ocean, significant for both its ecological role and commercial value. With climate change posing a threat to its survival, there is an urgent need to explore its adaptive capacity. Historical krill samples from the early 20th century offer a unique opportunity to examine changes in krill populations over time due to various pressures. This study evaluates two cost-effective genomic methods—low-coverage shotgun sequencing and exome capture—on 20 historical krill samples. The results included generating mitochondrial and nuclear ribosomal sequences, revealing genetic variability but no distinct population structure. Exome capture enhanced sequencing efficiency, enabling a deeper genetic analysis. This research aims to leverage historical collections for a better understanding of the biodiversity and adaptability of krill in the face of climate change.

Posterior fossa group A (PFA) ependymoma is a lethal brain cancer diagnosed in infants and young children. The lack of driver events in the PFA linear genome led us to search its 3D genome for characteristic features. Here, we reconstructed 3D genomes from diverse childhood tumor types and uncovered a global topology in PFA that is highly reminiscent of stem and progenitor cells in a variety of human tissues. A remarkable feature exclusively present in PFA are type B ultra long-range interactions in PFAs (TULIPs), regions separated by great distances along the linear genome that interact with each other in the 3D nuclear space with surprising strength. TULIPs occur in all PFA samples and recur at predictable genomic coordinates, and their formation is induced by expression of EZHIP. The universality of TULIPs across PFA samples suggests a conservation of molecular principles that could be exploited therapeutically.

Hybridization is essential for biodiversity and improving crop traits. Recent advances have successfully overcome the hybridization barrier between wheat and rice, two major cereals. Researchers produced allopolyploid hybrid zygotes in vitro, leading to mature plants with a unique genomic composition: wheat nuclear and cytoplasmic DNA paired with variable amounts (11%-47%) of rice mitochondrial DNA. This hybrid, termed OryzaWheat, can pass on rice mtDNA to future generations. Furthermore, a chromosome translocation was noted, where rice chromosome 1 integrated into wheat chromosome 6A. OryzaWheat opens new avenues for genetic resource utilization between the distinct subfamilies of wheat and rice.

Bacteriophages are promising alternatives to traditional antimicrobial treatment of bacterial infections. To further increase the potential of phages, efficient engineering methods are needed. This study investigates an approach to phage engineering based on phage rebooting and compares selected methods of assembly and rebooting of phage genomes. GG assembly of phage genomes and subsequent rebooting by cell-free transcription-translation reactions yielded the most efficient phage engineering and allowed production of a proof-of-concept T7 phage library of 1.8 × 107 phages. We obtained 7.5 × 106 different phages, demonstrating generation of large and diverse libraries suitable for high-throughput screening of mutant phenotypes. Implementing versatile and high-throughput phage engineering methods allows vastly accelerated and improved phage engineering, bringing us closer to applying effective phages to treat infections in the clinic.

Integral and interacting membrane proteins (IIMPs) are crucial biomolecules involved in various life processes, yet their characterization has been hindered by difficulties in purifying and labeling them. This study introduces a novel approach combining cell-free transcription-translation with quartz crystal microbalance with dissipation (TXTL-QCMD). This technique allows for the dynamic characterization of IIMP interactions with lipid bilayers without the need for purification or labeling. The method successfully reconstitutes known IIMP-membrane interactions, including the behavior of E. coli division proteins MinCDE. Additionally, it was applied to the Zorya anti-phage system, revealing specific interactions of the proteins ZorA and ZorB with bacterial membranes, while ZorE exhibited free diffusion. Overall, TXTL-QCMD demonstrates significant potential for exploring the diverse roles of IIMPs in biological systems.

Museum genomics provide an opportunity to investigate population demographics of extinct species, especially valuable when research prior to extinction was minimal. The Bachman’s warbler (Vermivora bachmanii) is hypothesized to have gone extinct due to loss of its specialized habitat. However, little is known about other potential contributing factors such as natural rarity or changes to connectivity following habitat fragmentation. We examined mitochondrial DNA (mtDNA) and genome-wide SNPs using specimens collected from breeding and migration sites across the range of the Bachman’s warbler. We found no signals of strong population structuring across the breeding range of Bachman’s warblers in both mtDNA and genome-wide SNPs. Thus, long-term population isolation did not appear to be a significant contributor to the extinction of the Bachman’s warbler. Instead, our findings support the theory that Bachman’s warblers underwent a rapid decline likely driven by habitat destruction, which may have been exacerbated by the natural rarity, habitat specificity and low genetic diversity of the species.

Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA’s presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage—all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.

Ligilactobacillus is a diverse genus among lactobacilli with phenotypes that reflect adaptation to various hosts. CRISPR-Cas systems are highly prevalent within lactobacilli, and Ligilactobacillus salivarius, the most abundant species of Ligilactobacillus, possesses both DNA- and RNA-targeting CRISPR-Cas systems. In this study, we explore the presence and functional properties of I-B, I-C, I-E, II-A, and III-A CRISPR-Cas systems in over 500 Ligilactobacillus genomes, emphasizing systems found in L. salivarius. We examined the I-E, II-A, and III-A CRISPR-Cas systems of two L. salivarius strains and observed occurrences of split cas genes and differences in CRISPR RNA maturation in native hosts. This prompted testing of the single Cas9 and multiprotein Cascade and Csm CRISPR-Cas effector complexes in a cell-free context to demonstrate the functionality of these systems. We also predicted self-targeting spacers within L. salivarius CRISPR-Cas systems and found that nearly a third of L. salivarius genomes possess unique self-targeting spacers that generally target elements other than prophages. With these two L. salivarius strains, we performed prophage induction coupled with RNA sequencing and discovered that the prophages residing within these strains are inducible and likely active elements, despite targeting by CRISPR-Cas systems. These findings deepen our comprehension of CRISPR-Cas systems in L. salivarius, further elucidating their relationship with associated prophages and providing a functional basis for the repurposing of these Cas effectors for bacterial manipulation.