ArticleBMC genomics2023
ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid RPE1 and HCT116 cells.
Article in BMC genomics, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
What it found
Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.
The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.
Who cites it
10 citing papers in PubMed, 12 citations in OpenAlex.
- Interfering with DNA repair pathways to enhance CRISPR-Cas9-mediated homology-directed repair in a chelicerate genetic model.iScience · 2026Article
- Zinc-redox crosstalk regulates proteostasis in the endoplasmic reticulum.Nature communications · 2026Article
- Strategies and considerations for the generation of ssDNA-Based HDR templates for CRISPR-based genome editing.BMC genomics · 2026Review
- Detection and Localization of Type II Membrane Proteins by Endogenous Gene Tagging and Confocal Microscopy.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Single-stranded HDR templates with truncated Cas12a-binding sequences improve knock-in efficiencies in primary human T cells.Molecular therapy. Nucleic acids · 2025Article
- Comparative analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging.Communications biology · 2025Article
- Precise Gene Knock-In Tools with Minimized Risk of DSBs: A Trend for Gene Manipulation.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2024Review
- Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing.Bio-protocol · 2024Article
- Genome editing in East African cichlids and tilapias: state-of-the-art and future directions.Open biology · 2023Review
- Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.Proceedings of the National Academy of Sciences of the United States of America · 2023Article
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
11 authors at 4 institutions in 2 countries.
Funding
Abstract
backgroundRecent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells.
resultsIn this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration.
conclusionsThese results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells.
Indexed as
Identifiers
What OpenQuestion holds
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.