ArticleMolecular therapy. Nucleic acids2017
Optimizing the DNA Donor Template for Homology-Directed Repair of Double-Strand Breaks.
Article in Molecular therapy. Nucleic acids, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 86 papers.
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Who cites it
86 citing papers in PubMed, 141 citations in OpenAlex.
- From Design to Single-Cell Cloning: A Complete RNP-Based CRISPR-Cas9 Protocol for Precision Gene Correction in Human iPSCs.Stem cell reviews and reports · 2026Article
- Highly frequent undesired insertional mutagenesis during Drosophila genome editing.PLoS genetics · 2026Article
- Oligonucleotide Synthesis Errors Are a Source of Untoward Variation in HDR-Mediated Gene Editing.Genes · 2026Article
- Prime Editing, CRISPR-Cas9, and NanoCas Genome Editing for Cancer Treatment.Molecular biotechnology · 2026Review
- Precise CRISPR-Mediated Editing of theInternational journal of molecular sciences · 2026Article
- Design and in silico validation of donor DNA for RNA-guided recombinase-mediated knockout of mstnb gene in Labeo rohita.PloS one · 2026Article
- CRISPR/Cas9-mediated gene targeting at BBM2 locus demonstrates HDR-assisted precise knock-in in banana cv. Grand Naine.Plant cell reports · 2025Article
- ONE-STEP tagging: a versatile method for rapid site-specific integration by simultaneous reagent delivery.Nucleic acids research · 2025Article
- A Gemini Virus-Derived Autonomously Replicating System for HDR-Mediated Genome Editing of thePlants (Basel, Switzerland) · 2025Article
- Gene Editing for Enhanced Swine Production: Current Advances and Prospects.Animals : an open access journal from MDPI · 2025Review
- A protocol for karyotyping and genetic editing of induced pluripotent stem cells with homology-directed repair mediated CRISPR/Cas9.Biology methods & protocols · 2025Article
- Gene Editing and Protein Tagging in the Oomycete Phytophthora infestans Using CRISPR-Cas12a.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Unlocking Fungal Potential: The CRISPR-Cas System as a Strategy for Secondary Metabolite Discovery.Journal of fungi (Basel, Switzerland) · 2024Review
- Fully defined NGN2 neuron protocol reveals diverse signatures of neuronal maturation.Cell reports methods · 2024Article
- Magnetic Nanoparticle-Assisted Non-Viral CRISPR-Cas9 for Enhanced Genome Editing to Treat Rett Syndrome.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2024Article
- Recent advances in CRISPR-Cas9-based genome insertion technologies.Molecular therapy. Nucleic acids · 2024Review
- CRISPR/Cas9 system and its applications in nervous system diseases.Genes & diseases · 2024Review
- Recent Research Trends in Stem Cells Using CRISPR/Cas-Based Genome Editing Methods.International journal of stem cells · 2024Review
- Precise base editing without unintended indels in human cells and mouse primary myoblasts.Experimental & molecular medicine · 2023Article
- A Review on Advanced CRISPR-Based Genome-Editing Tools: Base Editing and Prime Editing.Molecular biotechnology · 2023Review
26 more citing papers are in PubMed but not listed here.
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated proteins) technology enables rapid and precise genome editing at any desired genomic position in almost all cells and organisms. In this study, we analyzed the impact of different repair templates on the frequency of homology-directed repair (HDR) and non-homologous end joining (NHEJ). We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to quantify HDR and NHEJ events following transfection with Cas9, eight different guide RNAs, and a 1,000 bp donor template generated either as circular plasmid, as linearized plasmid with long 3' or 5' backbone overhang, or as PCR product. The sequence to be corrected was either centrally located (RS55), with a shorter 5' homologous region (RS37), or with a shorter 3' homologous region (RS73). Guide RNAs targeting the transcriptionally active strand (T5, T7) showed significantly higher NHEJ frequencies compared with guide RNAs targeting the transcriptionally inactive strand. HDR activity was highest when using the linearized plasmid with the short 5' backbone overhang and the RS37 design. The results demonstrate the importance of the design of the guide RNA and template DNA on the frequency of DNA repair events and, ultimately, on the outcome of treatment approaches using HDR.
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