CRISPR/Cas9-mediated Gene Editing in Murine Myoblasts

pSpCas9(BB)-2A-Puro-Gaa^c.1826 gRNA expression vectors and donor ssODNs were transfected into C2C12 murine myoblast cells (ATCC CRL-1772) using nucleofection-based transfection (Neon transfection system, Invitrogen). For nucleofection-based transfection, 3×10⁵ cells were resuspended in a reaction mixture containing 4.5 μg pSpCas9(BB)-2A-Puro-Gaa^c.1826 gRNA expression vector(s) and 450 nM ssODN. Cells were electroporated using the following parameters - Pulse voltage: 1650V, Pulse width: 10 ms, Pulse number: 3 – and plated onto duplicate wells of Matrigel-coated 6-well culture plates containing 2 mL culture media (DMEM + 10% FBS, 2 mM GlutaMax, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B) and maintained at 37 °C with 5%CO₂. 48 h post-transfection, cellular genomic DNA was extracted using QuickExtract DNA solution (Epicentre) and the Gaa^c.1826 target locus was PCR amplified and purified using DNA Clean & Concentrator (Zymo Research) and Sanger sequencing was performed (Retrogen). spCas9 nuclease activity and homology-directed repair (HDR) knock-in efficiency were determined by Tracking of Indels by Decomposition (TIDE)^{17 (link)} or Tracking of Insertion, Deletions, and Recombination events (TIDER)^{18 (link)} analysis of DNA sequence electropherogram files.