🧬 PubMed RNA Editing Daily Digest

Argonaute (Ago) proteins are programmable nucleases found in both eukaryotes and prokaryotes that use small nucleic acid guides to cleave complementary DNA or RNA targets. However, the limited catalytic efficiency and substrate versatility of currently characterized prokaryotic Argonautes (pAgos) have restricted their broader applications. Here, we characterized HthAgo, a pAgo from the thermophilic bacterium Hydrogenophilus thermoluteolus, and show that it possesses broad and precise nucleic acid cleavage activities. HthAgo efficiently cleaves DNA targets when loaded with 5'-phosphorylated DNA guides (5'P-gDNA), 5'-hydroxylated DNA guides (5'OH-gDNA), or 5'-phosphorylated RNA guides (5'P-gRNA), and also cleaves RNA targets when guided by 5'P-gDNA or 5'OH-gDNA. Remarkably, HthAgo maintains precise cleavage with both 5'P- and 5'OH-gDNA, with 5'P-guides directing canonical cleavage between guide positions 10 and 11, whereas 5'OH-guides shift cleavage exclusively to positions 11 and 12. Biochemical analyses showed that HthAgo remains active over a broad range of reaction conditions, displays optimal activity at 60-70 °C in the presence of Mn

Synthetic genomics is advancing from microbial toward multicellular organisms. However, current manual methods for DNA and genome assembly remain inadequate for the efficient, large-scale production of long DNA constructs. Here, we present Programmed DNA Assembly via Cas9 and Conjugative Transfer (PACT), a method that integrates a linear vector system, bacterial conjugation, and programmable Cas9-mediated cleavage to achieve highly efficient, iterative assembly of large DNA fragments. PACT enhances assembly efficiency by ~30-fold compared to conventional circular vector strategies, enabling one-step assembly of DNA up to 80 kb. We engineered four single guide-RNA-Marker donor cassettes to support iterative assembly workflows. PACT can utilize low-recombination Escherichia coli strains as hosts to efficiently assemble Arabidopsis thaliana mitochondrial genome with high repeat units. Integrated with an automated robotic platform, we developed an unattended, high-throughput pipeline (aPACT) toward large-scale parallel DNA assembly. Using aPACT, we successfully assembled three large DNA constructs: a 210 kb digital DNA, the designed chloroplast (120 kb) and mitochondrial (350 kb) genome of A. thaliana. This automated system offers a powerful tool for scalable assembly of large DNA molecules, accelerating synthetic genomics research toward complex multicellular organisms.

Reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome requires the first strand transfer that relies on a base-pairing interaction between the 3' R sequence of the genomic RNA (gRNA) and the r sequence of the strong-stop DNA (ssDNA). The annealing reaction between the cTAR hairpin of ssDNA and the TAR hairpin of gRNA plays an essential role in r-3' R pairing. Studies suggest that HIV-1 nucleocapsid protein (NC) has only a minor role in ssDNA synthesis but facilitates the annealing reaction by destabilizing the two complementary hairpins. We designed mutations to increase the stability of the cTAR DNA hairpin. These mutations should impair the NC-mediated annealing process, but not other viral processes. We investigated the effects of mutations in the annealing reaction, viral infectivity, and reverse transcription in infected cells. The mutations stabilizing the lower stem of the cTAR hairpin impair the annealing reaction, strongly reduce HIV-1 replication, and the amount of full-length ssDNA in infected cells. The

Genome editing has changed plant biology and accelerated crop improvement. CRISPR/Cas9 allows precise and efficient genetic changes in many species. Still, Cas9 has limits like PAM restrictions, off-target effects, and varying editing success. This led to new systems. Editors like Cas12a, CasΦ, CasMINI, and CasX offer more targeting options, can edit RNA, and work better with hard to edit plant genomes. Precision tools such as base editors and prime editors make precise changes by swapping nucleotides or adding small pieces without cutting both DNA strands. This improves accuracy. Beyond single tools, combined and step by step editing methods can be used for handling complex traits controlled by many genes. Using several methods like CRISPR knockouts, base and prime editing, epigenome editing and recombinase systems-breeders can improve traits while reducing unwanted side effects. Stepwise editing helps to test changes, confirm their effects, and improve entire biological pathways. Combining these approaches with AI-driven analysis, target prediction, and design optimization makes it easier to pick the best genes and edits for desired traits. These advanced editing methods are used to boost stress tolerance, fight diseases, improve nutrition, increase yields, and enhance quality after harvest. Despite progress, problems remain with how efficient edits can be made, delivering tools into plants, reliance on specific genotypes, unclear regulations, and acceptance by society. Looking ahead, joying genome editing with AI, fast breeding techniques help develop stronger, high yielding crops and support global food security.

Cephalosporin C (CPC)-derived antibiotics have played a vital role in improving human health and extending life expectancy.