🧬 PubMed RNA Editing Daily Digest

Adenosine Deaminase Acting on RNA (ADAR) is an enzyme that converts adenosine (A) to inosine (I) in RNA nucleotides, which can inhibit Dicer function during RNA interference, promoting viral replication and reducing the immune response. This research identified and characterized ADAR in the whiteleg shrimp, Penaeus vannamei, and investigated its role in regulating innate immunity during infection with the white spot syndrome virus (WSSV). The complete cDNA of P. vannamei ADAR (PvADAR) consisted of 2,073 nucleotides encoding 690 amino acids with a molecular weight of 77.5 kDa. PvADAR was highly expressed in hemocytes. Downregulation of PvADAR by injection of PvADAR-specific double-stranded RNA (dsPvADAR) at concentrations of 1 μg/g body weight resulted in upregulation of proPO, lysozyme, caspase-3, and argonaute-2 gene expression. Moreover, the total hemocyte count increased significantly from 6 to 72 h post dsPvADAR injection. The WSSV challenge was performed by immersion (10

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRSIPR-associated protein 9 (Cas9) has revolutionized genome editing with broad therapeutic applications, yet its repair patterns in vivo remain poorly understood. Here, we systematically profile CRISPR-Cas9 editing outcomes at 95 loci using our established CRISPR-Cas9/adeno-associated virus (AAV)9-single guide RNA (sgRNA) system in skeletal muscle stem cells (MuSCs). Through comprehensive characterization of the repair outcomes, our findings demonstrate that the general rules governing CRISPR-Cas9-mediated editing in vivo largely align with those observed in vitro. In addition to the anticipated small editing insertions or deletions (indels), such as microhomology-mediated end joining (MMEJ)-mediated deletions and non-homologous end joining (NHEJ)-mediated templated insertions, we uncover a prevalent occurrence of large on-target modifications, including large deletions (LDs) characterized by microhomology (MH) and large insertions (LIs). Notably, the LIs comprise not only exogenous AAV vector integrations but also endogenous genomic DNA fragments (Endo-LIs). Endo-LIs preferentially originate from active genomic regions, with their integration shaped by 3D chromatin architecture. By disrupting key components of the NHEJ and MMEJ repair pathways in vivo, we identify their distinct roles in regulating the large on-target modifications. Together, our work systematically profiles CRISPR-Cas9 repair outcomes in vivo and offers valuable guidance for improving the safety of CRISPR-Cas9-based gene therapies.

Adenosine deaminase acting on RNA 1 (ADAR1) deaminates adenosine to inosine in dsRNA. ADAR1 RNA editing marks cellular dsRNA as self, preventing aberrant activation of the antiviral dsRNA sensor MDA5. Adar Ifih1 (MDA5) or Adar Mavs double mutant pups escape Adar mutant aberrant interferon induction but die early. Here, we show that long-lived Adar Mavs Eif2ak2 (PKR) mice display a new residual defect: failure to maintain blood CD8+ T cells due to loss of an editing-independent function of ADAR1. Further dsRNA sensor-stripping in Adar Mavs Eif2ak2 Zbp1 mutants shows that loss of blood CD8+ T cells is not prevented. Challenging Adar+/- heterozygous or Adar Mavs Eif2ak2 mice with blood stage Plasmodium yoelii infection led to reduced parasitemia in Adar+/- mice by increased basal interferon. Unexpected reduced parasitemia in Adar Mavs Eif2ak2 mice is also likely due to interferon-related defects specific to hemopoietic cells, leading to altered populations of γδ T cells and other immune cells. The defects observed in this partially dsRNA sensor-stripped mouse suggest that different antiviral dsRNA sensors normally complement each other.