
Figure 2: Modification of Both HLA-C1 Alleles Using Multiple ssODN
CRISPR-Cas9 RNP combined with two ssODN DNA donor templates simultaneously edits both alleles of HLA-C1 in exon 2. One ssODN prevents Cas9 from re-cutting the first HLA-C1 allele by introducing a silent mutation. The other ssODN converts the second allele to HLA-C2 by substituting serine to asparagine at position 77 and asparagine to lysine at positions 77 and 80, respectively.
Figure 3: Successful, One-Step Biallelic Editing of iPSCs
The original DNA sequence is shown with the corresponding sgRNA below. The cleavage site in the DNA sequence is marked with a yellow arrow. ssODN1 contains a silent mutation highlighted in pink, while mutations in ssODN2 are highlighted in yellow. When analyzing bulk edited cells, each allele was found to have HDR efficiencies of approximately 24-28%. After Sanger sequencing analysis, one out of twenty-nine clones contained the expected modifications in both alleles.

Summary
- HLA heterozygous iPSCs were generated using CRISPR-Cas9 RNPs and ssODNs donor templates
- MaxCyte® electroporation of multiple ssODNs enabled highly efficient one-step biallelic engineering of iPSCs
- MaxCyte electroporation can be used to transiently transfect a variety of cell lines such as iPSCs and stem cells with high transfection efficiencies and cell viability
Reference:
1. 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)