ArticleThe CRISPR journal2022
Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors.
Article in The CRISPR journal, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 37 papers.
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Who cites it
37 citing papers in PubMed, 51 citations in OpenAlex.
- The use of homology-directed repair in a complex landscape of genome editors.Nature reviews. Molecular cell biology · 2026Article
- The Single-Stranded DNA Platform: A Potential Broad-Spectrum Vector for Editing Genes in Prokaryotes: ssDNA May Break Through the Difficulties of Genetic Manipulation on Non-Model Bacteria.BioEssays : news and reviews in molecular, cellular and developmental biology · 2026Review
- Cyanine-modified ssODNs enhance CRISPR-Cas9 HDR in stem cell embryo models via chromatin and chemical modulation.Nature communications · 2026Article
- Fully Modified SpyCas9 Guide RNAs Enable Robust Genome Editing In Cells and In Vivo.bioRxiv : the preprint server for biology · 2026Article
- A bulk cell heterozygous knock-in strategy for targeted protein degradation.bioRxiv : the preprint server for biology · 2026Article
- Ultra-large targeted DNA integrations in primary human cells.bioRxiv : the preprint server for biology · 2026Article
- Strategies and considerations for the generation of ssDNA-Based HDR templates for CRISPR-based genome editing.BMC genomics · 2026Review
- Orthogonal CRISPR systems for targeted integration and multiplex base editing enable nonviral engineering of allogeneic CAR-T cells.Molecular therapy : the journal of the American Society of Gene Therapy · 2025Article
- Nick-and-Digest Strategy for Programmable Circular ssDNA Production and Scalable DNA Origami Assembly.JACS Au · 2025Article
- Circular single stranded DNA potentiates non-viral gene insertion in hematopoietic stem and progenitor cells.Nature communications · 2025Article
- Modular Platform for Therapeutic Drug Delivery Using Trifunctional Bio-Orthogonal Macromolecular Conjugates.Bioconjugate chemistry · 2025Article
- The tumor microenvironment enhances the expression of cssDNA by modulating cell cycle signaling pathways via SKP2.Scientific reports · 2025Article
- Efficient non-viral immune cell engineering using circular single-stranded DNA-mediated genomic integration.Nature biotechnology · 2025Article
- Single-stranded HDR templates with truncated Cas12a-binding sequences improve knock-in efficiencies in primary human T cells.Molecular therapy. Nucleic acids · 2025Article
- Fluorescent reporter mouse models for genome editing: choices and challenges.Mammalian genome : official journal of the International Mammalian Genome Society · 2025Article
- Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration.Nature communications · 2025Article
- Fine-Tuning Homology-Directed Repair (HDR) for Precision Genome Editing: Current Strategies and Future Directions.International journal of molecular sciences · 2025Review
- CutIn: a ready-to-use construct for rapid generation of urgently needed transgenic cell lines in emerging infection research.Functional & integrative genomics · 2025Article
- From immune suppression to immunotherapy sensitization: the dual roles of circRNAs in cancer progression.Frontiers in immunology · 2025Review
- Rational Design of Enhanced Nme2Cas9 and Nme2bioRxiv : the preprint server for biology · 2024Article
Corrections and comments
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Authors and funding
16 authors at 2 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. In this study, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified traffic light reporter (TLR) system (TLR-multi-Cas variant 1 [MCV1]) that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knockins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knockins in mammalian cell lines.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.