ArticleScientific reports2023
CRISPR/Cas9-mediated targeted knock-in of large constructs using nocodazole and RNase HII.
Article in Scientific reports, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 17 citations in OpenAlex.
- Crosstalk-free optical indicator-actuator combinations for bidirectional optogenetics.npj biomedical innovations · 2026Review
- CRISPR-mediated precise large fragment insertion in zygotes enables rapid generation of humanized immunoglobulin heavy-chain mice.Immunity · 2026Article
- CRISPR Gene Tagging for Illuminating Endogenous Protein Dynamics.International journal of molecular sciences · 2026Review
- Label-free metabolic imaging monitors the fitness of chimeric antigen receptor T cells.Nature biomedical engineering · 2026Article
- Enhancing CRISPR Homology Directed Repair in IAL-PiD2 Insect Cells via Reagent Delivery Optimization and Cell Synchronization.Biochemical engineering journal · 2026Article
- Search-and-remove genome editing allows selection of cells by DNA sequence.Nature communications · 2025Article
- Rewriting the script: gene therapy and genome editing for von Willebrand Disease.Frontiers in genome editing · 2025Review
- Robust and cost-effective CRISPR/Cas9 gene editing of primary tumor B cells in Eµ-TCL1 model of chronic lymphocytic leukemia.HemaSphere · 2024Article
- CRISPR/Cas9-mediated base editors and their prospects for mitochondrial genome engineering.Gene therapy · 2024Review
- Increasing Knockin Efficiency in Mouse Zygotes by Transient Hypothermia.The CRISPR journal · 2024Article
- Improving homology-directed repair by small molecule agents for genetic engineering in unconventional yeast?-Learning from the engineering of mammalian systems.Microbial biotechnology · 2024Review
- Time and Cost-Effective Genome Editing Protocol for Simultaneous Caspase 8 Associated Protein 2 Gene Knock in/out in Chinese Hamster Ovary Cells Using CRISPR-Cas9 System.Iranian journal of biotechnology · 2024Article
- Modulation of cell cycle increases CRISPR-mediated homology-directed DNA repair.Cell & bioscience · 2023Article
- Editorial: Genetics of reproduction for livestock species.Frontiers in genetics · 2023Article
Corrections and comments
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Authors and funding
2 authors at 1 institution in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
On-target integration of large cassettes via homology-directed repair (HDR) has several applications. However, the HDR-mediated targeted knock-in suffered from low efficiency. In this study, we made several large plasmids (12.1-13.4 kb) which included the CRISPR/Cas9 system along with a puromycin transgene as part of the large DNA donor (5.3-7.1 kb insertion cassettes) and used them to evaluate their targeted integration efficiency into a transgenic murine embryonic fibroblast (MEF) cell line carrying a single copy of a Venus transgene. We established a detection assay by which HDR events could be discriminated from the error-prone non-homologous end-joining (NHEJ) events. Improving the plasmid quality could considerably leverage the cell toxicity impediment of large plasmids. The use of the TILD (targeted integration with linearized dsDNA) cassettes did not improve the HDR rate compared to the circular plasmids. However, the direct inclusion of nocodazole into the electroporation solution significantly improved the HDR rate. Also, simultaneous delivery of RNase HII and the donor plasmids into the electroporated cells considerably improved the HDR events. In conclusion, the results of this study showed that using cell synchronization reagents in the electroporation medium can efficiently induce HDR rate in the mammalian genome.
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