Molecular therapy. Nucleic acidsFeb 12, 2026

A new method to improve precise DNA repair in human stem cells using single-stranded donors

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Feb 13, 2026

Abstract

CRISPR-MiX significantly improved homology-directed repair (HDR) efficiency in human induced pluripotent stem cells (iPSCs).

Key design parameters of single-stranded oligodeoxynucleotide (ssODN) donors, such as homology arm symmetry and strand complementarity, strongly influence HDR outcomes.
Quantitative analysis indicated that both the target locus and ssODN design affect HDR efficiency.
CRISPR-MiX is a pooled ssODN-based method that enhances HDR efficiency without requiring selection.
The approach was successfully applied to introduce pathogenic variants into five genes associated with genetic cardiomyopathies.
This method offers a simple and versatile strategy for precise genome editing in iPSCs.

Simplified

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.

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A new method to improve precise DNA repair in human stem cells using single-stranded donors

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