🧬 PubMed RNA Editing Daily Digest

Engineered virus-like particles (eVLPs) are promising vehicles for transient delivery of gene editing agents. While extensive particle engineering has yielded efficient eVLPs, it remains underexplored whether engineering the cells used to produce eVLPs could further improve eVLP properties. We report an unbiased genome-wide screening approach to systematically investigate how genetic perturbations in producer cells influence eVLP production. This approach generates eVLPs loaded with guide RNAs that identify the genetic perturbation in the cell that produced a particular particle; the abundance of each guide RNA in eVLPs therefore reflects how the corresponding genetic perturbation influences eVLP production or cargo loading. We apply this approach to identify several genes that regulate eVLP cargo expression and loading into particles during the production process. Leveraging these insights, we engineer producer cells that support increased eVLP cargo packaging and a 2- to 9-fold increase in eVLP delivery potency across several cargo, particle, and target-cell types in cultured cells and in mice. Our findings suggest the potential of producer-cell engineering as a useful strategy for improving the utility of eVLPs and related delivery methods.

Post-transcriptional RNA modifications, such as N6-methyladenosine (m6A) methylation and adenosine to inosine (A-to-I) editing, are critical regulators of hematopoietic stem cell (HSC) self-renewal and differentiation, yet their precise contributions to malignant transformation are not fully elucidated. In this study, we uncovered the epitranscriptomic landscape caused by knockdown of genes from the methyltransferase (METTL)-family in hematopoietic stem and progenitor cells (HSPCs). We identified both converging and distinct effects of METTL3 and METTL14, known members of the m6A writer complex, as well as orphan gene METTL13. Amongst METTL-family members, only METTL13 transcription was increased following adenosine deaminase acting on RNA 1 (ADAR1) overexpression in HSPCs. This transcriptional pattern suggests that METTL13 may participate in biological programs that partially overlap with those controlled by the m6A writer complex and ADAR1, although any mechanistic relationship remains undefined. Knockdown of METTL13 altered the expression of multiple genes involved in oncogenic development in HSPCs. Furthermore, METTL13 expression was associated with a high-risk profile in pediatric T-cell acute lymphoblastic leukemia (T-ALL) and functional studies confirmed that METTL13 is required for T-ALL cell proliferation and survival both in vitro and in vivo. Collectively, our results identify METTL13 as a previously unrecognized regulator of leukemic transformation, independent of any presumed mechanistic interaction between RNA editing and m6A pathways.

AKT1 (Protein Kinase B alpha) is a serine/threonine kinase that plays a pivotal role in regulating various cellular processes. To elucidate the role of the AKT1 gene in signaling pathways, this study generated AKT1 knockout (KO) HT-1080 cells using the CRISPR/Cas9 system. Gene-editing efficiency was validated through Sanger DNA sequencing and insertion/deletion (InDel) analysis. Quantitative real-time PCR and Western blot analyses were performed to evaluate the expression levels of AKT1 mRNA and protein, as well as to examine the expression of AKT1 downstream effectors: mTOR, BCL-2, and FOXO1. The AKT1 single-guide RNA sequence was successfully cloned into the CRISPR/Cas9 vector, leading to the establishment of AKT1 KO cells. InDel analysis identified eight editing types, with two dominant populations. The expression levels of AKT1 mRNA and protein were significantly reduced in the KO cells. The expression levels of mTOR, BCL-2, and FOXO1 were significantly altered in the KO cells compared to normal cells. These findings highlight the impact of AKT1 disruption on signaling pathways and provide fundamental insights into the regulatory role of the AKT1 gene.