2. Academic Validation
  3. Enhanced Myogenesis by Silencing Myostatin with Nonviral Delivery of a dCas9 Ribonucleoprotein Complex

Enhanced Myogenesis by Silencing Myostatin with Nonviral Delivery of a dCas9 Ribonucleoprotein Complex

  • CRISPR J. 2022 Aug;5(4):598-608. doi: 10.1089/crispr.2022.0009.
Yinwei Chen 1 2,  Lia Banie 1,  Benjamin N Breyer 1,  Yan Tan 1,  Zhao Wang 1,  Feng Zhou 1,  Guifang Wang 1,  Guiting Lin 1,  Jihong Liu 3,  Lei S Qi 4 5,  Tom F Lue 1
Affiliations

Affiliations

  • 1 Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA; Stanford University, Stanford, California, USA.
  • 2 Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Stanford University, Stanford, California, USA.
  • 3 Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Stanford University, Stanford, California, USA.
  • 4 Department of Bioengineering, Stanford University, Stanford, California, USA; and Stanford University, Stanford, California, USA.
  • 5 ChEM-H, Stanford University, Stanford, California, USA.
PMID: 35758824 DOI: 10.1089/crispr.2022.0009
Abstract

Stress urinary incontinence (SUI) and pelvic floor disorder (PFD) are common conditions with limited treatment options in women worldwide. Regenerative therapy to restore urethral striated and pelvic floor muscles represents a valuable therapeutic approach. We aim to determine the CRISPR interference-mediated gene silencing effect of the nonviral delivery of nuclease-deactivated dCas9 ribonucleoprotein (RNP) complex on muscle regeneration at the cellular and molecular level. We designed four myostatin (MSTN)-targeting sgRNAs and transfected them into rat myoblast L6 cells together with the dCas9 protein. Myogenesis assay and immunofluorescence staining were performed to evaluate muscle differentiation, while CCK8 assay, cell cycle assay, and 5-ethynyl-2'-deoxyuridine staining were used to measure muscle proliferation. Reverse transcription-polymerase chain reaction and Western blotting were also performed to examine cellular signaling. Myogenic factors (including Myosin heavy chain, MSTN, myocardin, and serum response factor) increased significantly after day 5 during myogenesis. MSTN was efficiently silenced after transfecting the dCas9 RNP complex, which significantly promoted more myotube formation and a higher fusion index for L6 cells. In cellular signaling, MSTN repression enhanced the expression of MyoG and MyoD, phosphorylation of SMAD2, and the activity of Wnt1/GSK-3β/β-catenin pathway. Moreover, MSTN repression accelerated L6 cell growth with a higher cell proliferation index as well as a higher expression of cyclin D1 and cyclin E. Nonviral delivery of the dCas9 RNP complex significantly promoted myoblast differentiation and proliferation, providing a promising approach to improve muscle regeneration for SUI and PFD. Further characterization and validation of this approach in vivo are needed.

Figures
Products