ArticleScientific reports2021
Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK.
Article in Scientific reports, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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15 citing papers in PubMed, 26 citations in OpenAlex.
- SEED-Selection enables high-efficiency enrichment of primary T cells edited at multiple loci.Nature biotechnology · 2025Article
- Comparative analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging.Communications biology · 2025Article
- Dual inhibition of DNA-PK and Polϴ boosts precision of diverse prime editing systems.Nature communications · 2025Article
- Efficient nonviral integration of large transgenes into human T cells using Cas9-CLIPT.Molecular therapy. Methods & clinical development · 2025Article
- Magnetic Nanoparticle-Assisted Non-Viral CRISPR-Cas9 for Enhanced Genome Editing to Treat Rett Syndrome.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2024Article
- Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing.Bio-protocol · 2024Article
- CRISPR Ribonucleoprotein-Mediated Precise Editing of Multiple Genes in Porcine Fibroblasts.Animals : an open access journal from MDPI · 2024Article
- DNA-PK and the TRF2 iDDR inhibit MRN-initiated resection at leading-end telomeres.Nature structural & molecular biology · 2023Article
- Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing.Nature communications · 2023Article
- CRISPR/Cas9-mediated targeted knock-in of large constructs using nocodazole and RNase HII.Scientific reports · 2023Article
- Efficient simultaneous double DNA knock-in in murine embryonic stem cells by CRISPR/Cas9 ribonucleoprotein-mediated circular plasmid targeting for generating gene-manipulated mice.Scientific reports · 2022Article
- Genome editing.Scientific reports · 2022Article
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- Article
- Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing.International journal of molecular sciences · 2021Review
Corrections and comments
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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
CRISPR/Cas9-mediated homology-directed repair (HDR) is used for error-free targeted knock-in of foreign donor DNA. However, the low efficiency of HDR-mediated knock-in hinders establishment of knock-in clones. Double-strand breaks (DSBs) induced by CRISPR/Cas9 are preferentially repaired by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) before HDR can occur, thereby preventing HDR-mediated knock-in. NHEJ/MMEJ also cause random integrations, which give rise to false-positive knock-in events, or silently disrupt the genome. In this study, we optimized an HDR-mediated knock-in method for mouse embryonic stem cells (mESCs). We succeeded in improving efficiency of HDR-mediated knock-in of a plasmid donor while almost completely suppressing NHEJ/MMEJ-based integration by combining in vivo-linearization of the donor plasmid, transient knockdown of DNA polymerase θ, and chemical inhibition of DNA-dependent protein kinase (DNA-PK) by M3814. This method also dramatically improved the efficiency of biallelic knock-in; at the Rosa26a locus, 95% of HDR-mediated knock-in clones were biallelic. We designate this method BiPoD (Biallelic knock-in assisted by Pol θ and DNA-PK inhibition). BiPoD achieved simultaneous efficient biallelic knock-in into two loci. BiPoD, therefore, enables rapid and easy establishment of biallelic knock-in mESC lines.
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