Among the diverse prokaryotic adaptive immunity mechanisms, the Type III CRISPR-Cas systems are the most complex. The multisubunit Type III effectors recognize RNA targets complementary to CRISPR RNAs (crRNAs). Target recognition causes synthesis of cyclic oligoadenylates that activate downstream auxiliary effectors, which affect cell physiology in complex and poorly understood ways. Here, we studied the ability of III-A and III-B CRISPR-Cas subtypes from Thermus thermophilus to interfere with plasmid transformation. We find that for both systems, requirements for crRNA-target complementarity sufficient for interference depend on the target transcript abundance, with more abundant targets requiring shorter complementarity segments. This result and thermodynamic calculations indicate that Type III effectors bind their targets in a simple bimolecular reaction with more extensive crRNA-target base pairing compensating for lower target abundance. Since the targeted RNA used in our work is non-essential for either the host or the plasmid, the results also establish that a certain number of target-bound effector complexes must be present in the cell to interfere with plasmid establishment. For the more active III-A system, we determine the minimal length of RNA-duplex sufficient for interference and show that the position of this minimal duplex can vary within the effector. Finally, we show that the III-A immunity is dependent on the HD nuclease domain of the Cas10 subunit. Since this domain is absent from the III-B system the result implies that the T. thermophilus III-B system must elicit a more efficient cyclic oligoadenylate-dependent response to provide the immunity.

Extraction of high-quality microbial DNA remains a critical bottleneck in metagenomic research. Environmental samples often produce fragmented DNA and are prone to contaminations that interfere with downstream sequencing, while widely used commercial kits can be prohibitively expensive. Therefore, systematic evaluation of cost-effective alternatives is essential to support large-scale metagenomic studies. In this work, we benchmarked eight commercial DNA extraction kits from Magen, SkyGen, and Sileks against Qiagen reference kits. Four representative sample types were analyzed: freshwater, seafloor sediments, Pacific oyster (Magallana gigas) gut microbiome, and mammalian feces. DNA yield, integrity, purity, PCR inhibitor content, and eukaryotic DNA admixture were assessed. Microbial community composition, alpha diversity, reproducibility, and contamination (“kitome” and “splashome”) were further evaluated using 16S rRNA amplicon sequencing. We revealed that several alternative kits performed comparably or better than the Qiagen reference standard. Magen Soil and Magen Bacterial provided high yields and reproducibility, though the latter produced more fragmented DNA. SkyGen Stool excelled with host-associated samples, while Sileks Soil and Metagenomic kits preserved higher diversity in sediments. Magen Microbiome consistently underperformed. This study identifies multiple cost-effective DNA extraction strategies and provides practical guidance for selecting balanced DNA purification methods for different sample types.

Halisarca dujardinii is a marine sponge known for its ability to completely regenerate via cell reaggregation. Here we present the first chromosome-level genome assembly of H. dujardinii , generated using Oxford Nanopore long reads, Illumina short reads, and Hi-C data. The final assembly spans 226.5 Mbp and is organized into 21 chromosome-scale scaffolds, representing the full nuclear genome, with an assembly N50 of 10.1 Mbp. In addition, we report the complete mitochondrial genome for H. dujardinii , assembled as a circular molecule and annotated to contain 14 protein-coding genes. We provide a comprehensive genome annotation comprising 14,565 nuclear protein-coding genes, of which 85.6% are functionally annotated, along with repetitive elements and non-coding RNAs. Transcript models were refined using extensive bulk and single-cell RNA sequencing data, enabling accurate annotation of 3′ untranslated regions. This new genomic resource provides a valuable foundation for investigating the molecular basis of sponge regeneration, as well as for comparative studies of sponge evolution and marine biology.