Abstract
Muscle mass and area usually decrease with age, and this phenomenon is known as sarcopenia. This age-related atrophy correlates with insufficient levels of muscle cells differentiate and proliferate regulated by the TGF-β signaling pathway and the expression of E3s ubiquitin-protein ligase by the aged. Sarcopenia makes a huge impact on the aging society, because it has the characteristic of high incidence, extensive adverse effects and disease aggravation gradually. Guided by a single-guide RNA (sgRNA), Cas9 nuclease has been widely used in genome editing, opening up a new pathway for sarcopenia treatment. Here, we present two rAAV9 systems, pX601-AAV-CMV:SaCas9-U6:sgRNA and pX601-AAV-EF1α:SaCas9-tRNAGLN: sgRNA, which edited myostatin efficiently. By delivering the two rAAV–SaCas9 targets to myostatin via intramuscular injection of aged mice, an increase in body weight and an increase in the number and area of myofibers were observed. Knockout of myostatin led to TGF-β signaling pathway changes, and increased MyoD, Pax7 and MyoG protein levels and increased the number of satellite cells to improve muscle cells differentiation. Moreover, knockout of myostatin prevented the atrophy of muscle cells through reduced Murf1 and MAFbx protein levels. We found that both rAAV–SaCas9 systems had gene editing efficiency, reducing the expression of myostatin by affecting the relevant signaling pathways, thereby altering the physiological status. We showed that myostatin has an important role in activating skeletal muscle proliferation and inhibiting muscular atrophy during aging. Thus, we propose that knockout of myostatin using the rAAV9–SaCas9 system has significant therapeutic potential in sarcopenia.
Highlights
Cas9 is an RNA-guided endonuclease derived from the type II CRISPR–Cas adaptive immune system of bacterial adaptive immune system
We generated two versions of SaCas9 constructs with different singleguide RNA (sgRNA) promoters, U6 pro (~241 bp) and tRNA GLN pro (~72 bp), and different SaCas9 promoters, CMV pro (~584 bp) and EF1α pro (~212 bp)
The rapid development and application of CRISPR/Cas9 technology has led to a number of studies on its efficacy and specificity, most of these studies have focused on SpCas9
Summary
Cas is an RNA-guided endonuclease derived from the type II CRISPR–Cas adaptive immune system of bacterial adaptive immune system. For the efficient delivery of CRISPR materials into cells in vivo, Wei et al [4] reconstructed the new vector lentiSaCRISPR v2, which was the original lentiCRISPR v2 that targets SpCas9 [5] with the Cas and sgRNA cassettes switched to make it adaptable for SaCas targeting. We generated two versions of SaCas constructs with different sgRNA promoters, U6 pro (~241 bp) and tRNA GLN pro (~72 bp), and different SaCas promoters, CMV pro (~584 bp) and EF1α pro (~212 bp). These promoters are reported to be able to effectively drive the expression of Cas and can be used to efficiently edit genomic DNA in vitro and in vivo
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