One of the most efficient and powerful genome-editing instruments, CRISPR-Cas9 represents a high promise in treating disease and examining the genome. Nevertheless, its clinical translation is limited by off-target effects that cause harmful genomic mutations and potentially fatal safety issues. This review provides a thorough overview of the current state of knowledge on off-target causes, detection and prediction methods, and mitigation strategies. MAIN REASONSThese include mismatch tolerance of Cas9-gRNA and sustained expression associated with some delivery methods. Finally, the detection and prediction tools based on representative approaches such as GUIDE-seq, CIRCLE-seq, Cas-OFFinder and DeepCRISPR are evaluated and their advantages and limitations are listed. The mitigation strategies such as sgRNA engineering, Cas9 protein engineering and transient delivery systems are also discussed. The study argues that the data mentioned above only measures an important first step, and that the realization of clinically relevant detection models that reliably mimic in vivo conditions is critical to screening safety. This review integrates these dimensions, so as to offer a clear framework for controlling when, where and how. Integrating these dimensions allows this review to provide a clear framework for controlling when, where and how CRISPR-Cas9-mediated alterations occur afford systemic implementation strategies that improve the safety of CRISPR-Cas9 applications.
Yanbing Xu· Theoretical and Natural Scie...· 0 citations
Mitochondrial dysfunction is an important cause of sarcopenia, and TWEAK/Fn14, as one of the major muscle wasting cytokines, its role in the development of sarcopenia by regulating mitochondrial biogenesis remains unclear. Expression of TWEAK in old and young mice was both detected. TWEAK was silenced in C2C12 myocytes using lentiviral vectors. Immunofluorescence, western blot, real-time polymerase chain reaction (RT-PCR), and ELISA were enrolled to analyze the effects of TWEAK on myotube size, mitochondrial content, mitochondrial ROS, and inflammatory factors. Additionally, aged mice received two injections of AAV9 vectors at 15 and 17 months of age. Upon reaching 18 months of age, the effects of TWEAK knockdown on grip strength, muscle mass, and gastrocnemius muscle indices were evaluated. TWEAK/Fn14 expression was significantly increased in old mice (
p
< 0.001). Compared with young controls, old mice exhibited a significant decrease in grip strength (
p
< 0.001) and a significant increase in lean mass (
p
< 0.05), whereas no significant difference was observed in fat content. In DEX-treated C2C12 myotubes, TWEAK knockdown significantly increased myotube diameter, enhanced ATP content and mitochondrial quantity, upregulated protein expression of SIRT1, PGC-1α, and p-AMPK, and inhibited mitochondrial ROS, Ca
2+
levels, p-p38 expression, and the secretion of inflammatory cytokines (TNF-α, IL-1β, IL-6, and iNOS). In aged mice, TWEAK knockdown did not significantly alter forelimb grip strength or lean mass, but significantly increased fat mass (
p
< 0.05). Mechanistically, TWEAK knockdown promoted AMPK signaling, inhibited p38 MAPK activation, enhanced mitochondrial biogenesis, and reduced serum levels of IL-1β, IL-6, and iNOS (
p
< 0.05), whereas serum TNF-α levels showed no significant difference. TWEAK knockdown attenuates age-related skeletal muscle mass loss and improves mitochondrial biogenesis in skeletal muscle, accompanied by modulated inflammatory factor release and altered AMPK-p38 MAPK signaling activity. However, no significant improvement in forelimb grip strength was observed in the in vivo experiment. These findings indicate that TWEAK suppression may represent a promising strategy for preserving muscle mass and metabolic homeostasis during aging, though its capacity to fully restore functional capacity requires further investigation.
Zhuoya Maimaitiwusiman, Saiyare Xuekelati, Anyan Wang et al.· Scientific Reports· 0 citations