ArticleNucleic acids research2024
Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9.
Article in Nucleic acids research, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed.
- Chromatin Modifications Orchestrate DNA Repair Pathway Choice: Epigenetics Strategies for Enhancing CRISPR-Cas9-Mediated Knock-In Efficiency.Cell proliferation · 2026Review
- Industry Perspective on Translational and Clinical Pharmacology Aspects of Viral-based Gene Therapies and Vaccines-Key Considerations and Learnings From Approved Products.The AAPS journal · 2026Review
- Cyanine-modified ssODNs enhance CRISPR-Cas9 HDR in stem cell embryo models via chromatin and chemical modulation.Nature communications · 2026Article
- The cellular landscape of i-motifs: genomic insights, methodological challenges, and the road ahead.Genome biology · 2026Review
- Predicting Single-Stranded DNA Oligonucleotides 3D Structures: An Open Issue.Computational and structural biotechnology journal · 2026Article
- Circular single stranded DNA potentiates non-viral gene insertion in hematopoietic stem and progenitor cells.Nature communications · 2025Article
- Single-stranded HDR templates with truncated Cas12a-binding sequences improve knock-in efficiencies in primary human T cells.Molecular therapy. Nucleic acids · 2025Article
- Progress of personalized medicine of cystic fibrosis in the times of efficient CFTR modulators.Molecular and cellular pediatrics · 2025Review
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13 authors.
Funding
Abstract
Single-stranded DNA (ssDNA) templates along with Cas9 have been used for knocking-in exogenous sequences in the genome but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 12-19% of internal bases, which we denote as enhanced ssDNA (esDNA), improve knock-in (KI) by 2-3-fold compared to end-modified ssDNA in airway basal stem cells (ABCs), CD34 + hematopoietic cells (CD34 + cells), T-cells and endothelial cells. Over 50% of alleles showed KI in three clinically relevant loci (CFTR, HBB and CCR5) in ABCs using esDNA and up to 70% of alleles showed KI in the HBB locus in CD34 + cells in the presence of a DNA-PKcs inhibitor. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. The esDNA templates did not improve KI in induced pluripotent stem cells (iPSCs). This may be due to the absence of the nuclease TREX1 in iPSCs. Indeed, knocking out TREX1 in other cells improved KI using unmodified ssDNA. esDNA can be used to modify 20-30 bp regions in primary cells for therapeutic applications and biological modeling. The use of this approach for gene length insertions will require new methods to produce long chemically modified ssDNA in scalable quantities.
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