COClinical Orthopaedics3083-15393083-1520Bio-Byword Scientific Publishing Pty. Ltd.10.26689/co.v1i1.15875ArticleCRISPR/Cas9 Editing of miR-146a Suppresses Immobilization-induced Muscle Atrophyhttps://artdesignp.com/journal/CO/1/1/10.26689/co.v1i1.15875LiLu,GaoXiang,WangHui,QiaoLi,XuXiao,YanYuxin,YaoJiahao,BaiJiayu,XiaZhirong,MiaoShuai2026112026-08-12Immobilization-induced muscle wasting is notoriously hard to counter pharmacologically. We tested whether CRISPR-mediated knockdown of miR-146a could offer protection and asked what might explain such an effect. Four sgRNAs were designed against the mouse miR-146a precursor; the best performer (lentiCRISPR miR-146a-3#), which gave ~80% cleavage in T7EI assays, was packaged into lentivirus. Forty C57BL/6 mice were randomly assigned to receive either this editing construct or a control vector, then underwent sham surgery or hindlimb immobilization. Western blotting confirmed Cas9 expression, and qPCR verified miR-146a reduction. In immobilized mice, miR-146a rose markedly, accompanied by elevated Atrogin-1 and MuRF-1 mRNAs, reduced myofiber cross-sectional area, and diminished p-mTOR and p-Akt signals. By contrast, animals given the miR-146a-editing virus showed lower atrogene expression, larger fibers, and restored mTOR/Akt phosphorylation. These results suggest that CRISPR-based miR-146a ablation mitigates disuse atrophy, and this benefit appears to be linked to reactivation of the mTOR/Akt anabolic pathway.

Lopes J, Russell DM, Whitwell J, et al., 1982, Skeletal Muscle Function in Malnutrition, Am J Clin Nutr, 36(4): 602-610.

English C, Mclennan H, Thoirs K, et al., 2010, Loss of Skeletal Muscle Mass after Stroke: A Systematic Review, Int J Stroke, 5(5): 395-402.

Topp R, Ditmyer M, King K, et al., 2002, The Effect of Bed Rest and Potential of Prehabilitation on Patients in the Intensive Care Unit, AACN Clin Issues, 13(2): 263-276.

Booth FW, Seider MJ, 1979, Recovery of Skeletal Muscle after 3 Mo of Hindlimb Immobilization in Rats, J Appl Physiol Respir Environ Exerc Physiol, 47(2): 435-439.

Tischler ME, Rosenberg S, Satarug S, et al., 1990, Different Mechanisms of Increased Proteolysis in Atrophy Induced by Denervation or Unweighting of Rat Soleus Muscle, Metabolism, 39(7): 756-763.

Jaspers SR, Tischler ME, 1984, Atrophy and Growth Failure of Rat Hindlimb Muscles in Tail-cast Suspension, J Appl Physiol Respir Environ Exerc Physiol, 57(5): 1472-1479.

Ivey KN, Srivastava D. microRNAs as Developmental Regulators, 2015, Cold Spring Harb Perspect Biol, 7(7): a008144.

Von Haehling S, Ebner N, Dos Santos M R, et al., 2017, Muscle Wasting and Cachexia in Heart Failure: Mechanisms and Therapies, Nat Rev Cardiol, 14(6): 323-341.

Bei Y, Xiao J, MicroRNAs in Muscle Wasting and Cachexia Induced by Heart Failure, 2017, Nat Rev Cardiol, 14(9): 566.

Horak M, Novak J, Bienertova-Vasku J, 2016, Muscle-specific microRNAs in Skeletal Muscle Development, Dev Biol, 410(1): 1-13.

Rau CS, Jeng JC, Jeng SF, et al., 2010, Entrapment Neuropathy Results in Different microRNA Expression Patterns from Denervation Injury in Rats, BMC Musculoskelet Disord, 11: 181.

Nelson CE, Hakim CH, Ousterout DG, et al., 2016, In Vivo Genome Editing Improves Muscle Function in a Mouse Model of Duchenne Muscular Dystrophy, Science, 351(6271): 403-407.

Wei Y, Chen Y, Qiu Y, et al., 2016, Prevention of Muscle Wasting by CRISPR/Cas9-mediated Disruption of Myostatin In Vivo, Mol Ther, 24(11): 1889-1891.

Xu L, Park KH, Zhao L, et al., 2016, CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice, Mol Ther, 24(3): 564-569.

Lataniotis L, Albrecht A, Kok FO, et al., 2017, CRISPR/Cas9 Editing Reveals Novel Mechanisms of Clustered microRNA Regulation and Function, Sci Rep, 7(1): 8585.

Huo W, Zhao G, Yin J, et al., 2017, Lentiviral CRISPR/Cas9 Vector Mediated miR-21 Gene Editing Inhibits the Epithelial to Mesenchymal Transition in Ovarian Cancer Cells, J Cancer, 8(1): 57-64.

Chang H, Yi B, Ma R, et al., 2016, CRISPR/cas9, A Novel Genomic Tool to Knock Down microRNA in Vitro and in Vivo, Sci Rep, 6: 22312.

Li J, Chan MC, Yu Y, et al., 2017, miR-29b Contributes to Multiple Types of Muscle Atrophy, Nat Commun, 8: 15201.

Liu X, Xiao J, Zhu H, et al., 2015, miR-222 is Necessary for Exercise-induced Cardiac Growth and Protects Against Pathological Cardiac Remodeling, Cell Metab, 21(4): 584-595.

Friedmann T, Roblin R, Gene Therapy for Human Genetic Disease?, Science, 1972, 175(4025): 949-955.

Nelson CE, Robinson-Hamm JN, Gersbach CA., 2017, Genome Engineering: A New Approach to Gene Therapy for Neuromuscular Disorders, Nat Rev Neurol, 13(11): 647-661.

Egerman MA, Glass DJ, Signaling Pathways Controlling Skeletal Muscle Mass, 2014, Crit Rev Biochem Mol Biol, 49(1): 59-68.

Kok K, Geering B, Vanhaesebroeck B, 2009, Regulation of Phosphoinositide 3-kinase Expression in Health and Disease, Trends Biochem Sci, 34(3): 115-127.

Yoon MS, 2017, mTOR as a Key Regulator in Maintaining Skeletal Muscle Mass, Front Physiol, 8: 788.