Highly Efficient Homozygous Correction of the DYSF Gene in Miyoshi Myopathy Patient iPSCs by ssODN-Mediated Knockin
Human induced pluripotent stem cells (iPSCs) play an important role in disease modeling and drug screening, and have tremendous potential for regenerative medicine. Over half all reported pathogenic mutations are caused by single-nucleotide polymorphisms (SNP); as an example, Miyoshi myopathy is a congenital muscle wasting disease caused by a mutation in the dysferlin (DYSF) gene. The correction of such mutations in iPSCs is critical to generating corrected isogenic clones for basic research and for autologous use in clinical applications. However, low efficiencies of correction (<5%) result in time-consuming and laborious screening of many clones to establish an iPSC line. Hotta et al., used the MaxCyte® platform to electroporate 100 bp single stranded oligodeoxynucleotides (ssODN) and CRISPR-Cas9 RNP achieving greater than 70% homozygous SNP correction in the DYSF gene of Miyoshi myopathy patient iPSCs.
- Footprint-free homozygous correction of a SNP mutation was achieved with over 70% efficiency in the absence of selection.
- MaxCyte electroporation enabled higher cell survival compared to an alternative electroporation platform.
- Higher cell viability post electroporation supports higher HDR frequencies in the final cell population.
- Higher HDR efficiency results in lower cost, less labor and shortened timelines.
- MaxCyte electroporation can be used to transiently transfect a variety cell lines such as iPSCs and stem cells with high transfection efficiencies and cell viability.
- Kagita A, Lung MSY, Xu H, et al. Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA Ribonucleoprotein. Stem Cell Reports. 2021;16(4):985-996. doi:10.1016/j.stemcr.2021.02.013
This content was adapted from Kagita et al. 2021 under the Creative Commons license Attribution 4.0 International (CC BY 4.0)