🧬 PubMed RNA Editing Daily Digest

SCN8A encodes the voltage-gated sodium channel Nav1.6, which plays a key role in facilitating neuronal excitability. Mutations in SCN8A, particularly gain-of-function variants, cause SCN8A developmental and epileptic encephalopathy (DEE), a severe epilepsy syndrome characterized by seizures, cognitive dysfunction, movement disorders, and sudden unexpected death in epilepsy (SUDEP). The recurrent SCN8A variant R1872W impairs channel inactivation, causing neuronal hyperexcitability and seizures. Current treatments, including antiseizure medications, are often ineffective for patients with SCN8A DEE, highlighting the need for targeted therapies. We employed base editing to correct the R1872W SCN8A variant. An adenine base editor and guide RNA (SCN8A-ABE) were packaged within dual PhP.eB-adeno-associated viruses (AAVs) and administered to R1872W mice at P2. SCN8A-ABE significantly increased survival of mice expressing R1872W and either reduced seizure incidence and severity or eliminated seizure occurrence. Electrophysiological recordings revealed a rescue of seizure-associated neuronal hyperexcitability and suppression of the pathogenic persistent sodium current (INaP) in treated mice. Comorbidities, including diminished mobility and anxiety-like behaviors, were improved by SCN8A-ABE. These effects were achieved by a 32% absolute reduction in mutant transcripts, accompanied by conversion to SCN8A WT transcripts. Our findings demonstrate base editing as an effective targeted therapeutic approach for SCN8A DEEs by addressing the underlying genetic cause.

Interspecies chimeras serve as valuable models for studying mammalian development and advancing strategies for organ generation. However, xenogeneic barriers-such as mismatched developmental timing, cell adhesion differences, and intercellular competition-restrict the contribution of human pluripotent stem cells (hPSCs) to non-human embryos. Here, we report that Cas13 collateral RNA cleavage can be harnessed to selectively attenuate host cell proliferation to boost donor cell chimerism. In mouse epiblast stem cells (mEpiSCs), Cas13 activation degraded target RNAs and bystander transcripts, producing a reversible decrease in cell proliferation while preserving pluripotency. In co-culture, selective attenuation of mEpiSCs enhanced the competitiveness and survival of wild-type mEpiSCs and hPSCs in both intra- and interspecies settings. In vivo, blastocysts engineered with inducible Cas13 (iCas13) showed increased donor cell integration in chimeric embryos and adult tissues. Together, these findings demonstrate a generalizable strategy to modulate donor-host dynamics and mitigate xenogeneic barriers to interspecies organogenesis.

The growing success of RNA-based therapeutics has emphasized the need for precise and programmable RNA synthesis platforms. T7 RNA polymerase (T7RP) is widely utilized for in vitro transcription; however, most existing regulatory strategies rely on auxiliary proteins or chemical modulators. Here, we investigated whether transcription can be regulated solely through nucleic acid sequences. Specifically, we evaluated the effects of single-stranded DNA flap sequences appended to the 3' end of the non-template strand of the T7 promoter, termed the flap promoter, on transcriptional efficiency. Remarkably, we observed a sequence-dependent inhibitory effect, wherein flaps enriched in pyrimidines (cytosine and thymine) significantly suppressed T7RP-mediated transcription. Leveraging this intrinsic sequence preference, we developed two novel transcription control platforms, D-FIT (DNAzyme-mediated Flap promoter Induced Transcription control) and M-FIT (MNAzyme-mediated Flap promoter Induced Transcription control) that enable precise regulation of T7RP activity without the need for auxiliary proteins or chemical agents. These findings uncover a previously unrecognized sequence-specific regulatory mechanism of T7RP and establish a new framework for the rational design of programmable RNA synthesis systems, with broad potential applications in RNA therapeutics and diagnostics.

The CRISPR/Cas9 system revolutionizes genome engineering, yet optimizing the stability and expression levels of single-guide RNA (sgRNA) is crucial for achieving more effective gene regulation. Transfer RNAs (tRNA), known for their inherent stability, present a valuable solution. In this study, we developed a chimeric tRNA-sgRNA (tgRNA) by integrating sgRNA into the anticodon stem of a Sephadex aptamer-human HBV ε tRNA (SeptRNA) scaffold, resulting in the formation of SeptgRNA. When applied to target the E. coli ampC and ompA genes, SeptgRNA exhibited significantly increased accumulation compared to conventional sgRNAs. To overcome potential steric hindrance from the tRNA scaffold, we utilized CRISPR interference (CRISPRi) by co-expressing SeptgRNA with deactivated Cas9 (dCas9), which effectively suppressed DNA transcription. This approach demonstrated superior gene expression suppression compared to traditional sgRNA-based CRISPRi. Molecular docking and molecular dynamics simulations revealed that the SeptRNA scaffold stabilizes the sgRNA stem-loop architecture and enhances the stability of the dCas9-tgRNA-DNA ternary complex. Our findings provide proof-of-concept for the use of chimeric tgRNAs in gene knockdown, highlighting their potential for increased expression levels and improved stability. This study advances the CRISPR/Cas9 toolkit and underscores the versatility of tRNA scaffolds in genetic engineering applications.