CRISPR-MiX: A pooled single-stranded donor strategy to enhance HDR efficiency in human iPSCs
Summary
CRISPR-Cas9 is widely used to model genetic disorders by introducing or correcting disease-associated mutations in induced pluripotent stem cells (iPSCs) through homology-directed repair (HDR). However, HDR efficiency in iPSCs remains low and is highly dependent on the target locus. Here, we developed CRISPR-MiX, an improved protocol to enhance HDR efficiency in human iPSCs. Using a GFP-to-BFP reporter system, we identified key single-stranded oligodeoxynucleotide (ssODN) donor design parameters, including homology arm symmetry, CRISPR/Cas-blocking mutations, and strand complementarity, which significantly influence HDR outcomes. We applied this approach to introduce pathogenic variants into five genes related to genetic cardiomyopathies. Quantitative analysis of HDR events showed that both the target locus and ssODN design strongly affect HDR efficiency. To address the locus- and design-specific limitations, we established CRISPR-MiX, a pooled ssODN-based method for scarless genome editing using ribonucleoproteins (RNPs) that does not require selection. CRISPR-MiX consistently improved HDR efficiency across multiple loci. This strategy offers a simple, robust, and versatile approach for precise genome engineering in iPSCs, supporting broad applications in disease modeling and functional genomics. © 2026 The Authors.
| Authors | Baum R, Telugu N, Bruyneel AAN, Kay M, Nair P, Perea-Gil I, Termglinchan V, Bharucha N, Lee E, Mercola M, Diecke S, Karakikes I |
|---|---|
| Journal | Molecular therapy. Nucleic acids |
| Publication Date | 2026 Mar 12;37(1):102820 |
| PubMed | 41674724 |
| PubMed Central | PMC12887267 |
| DOI | 10.1016/j.omtn.2025.102820 |