Necroptosis is implicated in the pathogenesis of ischemic stroke. However, the mechanism underlying the sequential recruitment of receptor-interacting protein kinase 1 (RIPK1) and N-ethylmaleimide-sensitive fusion ATPase (NSF) in initiating necroptosis remains poorly understood, and the role of NSF in ischemic stroke is a subject of controversy. Here, we utilized a recently emerging RNA-targeting CRISPR system known as CasRx, delivered by AAVs, to knockdown Ripk1 mRNA and Nsf mRNA around the ischemic brain tissue. This approach resulted in a reduction in infarct and edema volume, as well as an improvement in neurological deficits assessed by Bederson score, RotaRod test, and Adhesive removal test, which were achieved by RIPK1/receptor-interacting protein kinase 3/mixed lineage kinase domain-like protein signaling pathway involved in neuronal necroptosis. In conclusion, the downregulation of Ripk1 mRNA and Nsf mRNA mediated by CRISPR-CasRx holds promise for future therapeutic applications aimed at ameliorating cerebral lesions and neurological deficits following the ischemic stroke.

CasRx, a recently discovered member of the type VI CRISPR system with minimum size, offers a new approach for RNA manipulation with high efficiency and specificity in prokaryotes and eukaryotes. However, in vivo studies of functional recovery using the CasRx system have not been well characterized. Here, we sought to establish an adeno-associated virus (AAV)-CasRx-guide RNA (gRNA) system for the specific knockdown of Htra2 transcript to protect mice from aminoglycosides-induced hearing loss. For the study, we verified an optimized gRNA in vitro, which was packaged into a single AAV with CasRx, and injected the packaged AAV into mice with hearing loss induced by neomycin and auditory functions investigated by auditory brainstem response tests. Upon using the AAV-CasRx-gRNA system, we found the knockdown of Htra2 transcript led to less cochlear hair cell loss and improved auditory function, with low off-target and adverse side effects. Additionally, the decrease in Htra2 significantly inhibits mRNA expression of Casp3 and Casp9. In conclusion, the AAV-CasRx-gRNA-mediated knockdown of Htra2 transcript in mice has been proved effective and safe for preventing hearing loss induced by aminoglycosides and, thus, represents a promising genetic approach for the future clinical applications for treating non-inherited hearing loss.

The RNA editing tool CRISPR-CasRx has provided a platform for a range of transcriptome analysis tools and therapeutic approaches with its broad efficacy and high specificity. To enable the application of CasRx in vivo, we established a Credependent CasRx knock-in mouse. Using these mice, we specifically knocked down the expression of Meis1 and Hoxb13 in cardiomyocytes, which induced cardiac regeneration after myocardial infarction. We also knocked down the lncRNA Mhrt in cardiomyocytes with the CasRx knock-in mice, causing hypertrophic cardiomyopathy. In summary, we generated a Credependent CasRx knock-in mouse that can efficiently knock down coding gene and lncRNA expression in specific somatic cells. This in vivo CRISPR-CasRx system is promising for gene function research and disease modeling.

In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo.