Related Experiment Video
Updated: Oct 26, 2026
Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing
Published on: June 16, 2022
A DNA-free CRISPR-Cas9 ribonucleoprotein electroporation protocol for efficient knockout generation in cardiac and
Babak Nami1, Yuhe Xiao2, Gurpreet Kaur2
1University of New Brunswick, Saint John, New Brunswick, Canada; Dynoma Biosciences Inc, Saint John, New Brunswick, Canada.
Background:
Efficient, reproducible gene knockout in cardiomyocyte-related cell models is critical for cardiovascular research but remains challenging because of cell-type-specific sensitivity to genome-editing workflows.
Methods:
We developed an optimized CRISPR-Cas9 genome-editing protocol using electroporation of ribonucleoprotein (RNP) complexes to generate knockout lines in cardiac and skeletal muscle cells. We also provide practical guidance for gRNA design, genotyping, and clonal line generation. Using human AC16 cardiomyocyte-like cells and rodent muscle-derived cell lines (C2C12, L6, and H9C2), we optimized electroporation conditions to maximize editing efficiency while maintaining viability.
Results:
SpCas9-single-guide RNA (sgRNA) RNPs targeting the lipid-trafficking regulator SAR1B/Sar1b produced high-frequency frameshift indels and marked loss of target protein expression in all models. A composite knockout score that combines editing efficiency and cell survival identified optimal electroporation settings of 1400 V, 10 ms, and 3 pulses for AC16 cells, and 1500 V, 10 ms, and 3 pulses for C2C12, L6, and H9C2 cells.
Conclusions:
This protocol combines rational sgRNA design, chemically modified sgRNAs for efficient RNP assembly, and standardized genotyping to improve reproducibility. The framework is transferable to other cardiomyocyte and muscle-derived cell types and supports both pthe olyclonal and monoclonal CRISPR-Cas9 knockout approaches.
