Comparative analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging
Abstract CRISPR-mediated endogenous tagging is a powerful tool in biological research. Inhibiting the non-homologous end joining (NHEJ) pathway has been shown to improve the low efficiency of accurate knock-in via homology-directed repair (HDR). However, the influence of alternative double-stranded...
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| Format: | Article |
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Nature Portfolio
2025-05-01
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| Series: | Communications Biology |
| Online Access: | https://doi.org/10.1038/s42003-025-08187-5 |
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| author | Chiharu Tei Shoji Hata Akira Mabuchi Shotaro Okuda Kei K. Ito Mariya Genova Masamitsu Fukuyama Shohei Yamamoto Takumi Chinen Atsushi Toyoda Daiju Kitagawa |
| author_facet | Chiharu Tei Shoji Hata Akira Mabuchi Shotaro Okuda Kei K. Ito Mariya Genova Masamitsu Fukuyama Shohei Yamamoto Takumi Chinen Atsushi Toyoda Daiju Kitagawa |
| author_sort | Chiharu Tei |
| collection | DOAJ |
| description | Abstract CRISPR-mediated endogenous tagging is a powerful tool in biological research. Inhibiting the non-homologous end joining (NHEJ) pathway has been shown to improve the low efficiency of accurate knock-in via homology-directed repair (HDR). However, the influence of alternative double-stranded break (DSB) repair pathways on knock-in remains to be fully explored. In this study, our long-read amplicon sequencing analysis reveals various patterns of imprecise repair in CRISPR-mediated knock-in, even with NHEJ inhibition. Further suppressing either microhomology-mediated end joining (MMEJ) or single-strand annealing (SSA) reduces nucleotide deletions around the cut site, thereby elevating knock-in accuracy. Additionally, imprecise donor integration is reduced by inhibiting SSA, but not MMEJ. Particularly, SSA suppression reduced asymmetric HDR, a specific imprecise integration pattern, which we further confirm using a novel reporter system. These findings demonstrate the complex interplay of multiple DSB repair pathways in CRISPR-mediated knock-in and offer novel strategies, including SSA pathway targeting, to improve precise gene editing efficiency. |
| format | Article |
| id | doaj-art-b6eea10a628e497987d7685b2e671805 |
| institution | DOAJ |
| issn | 2399-3642 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Biology |
| spelling | doaj-art-b6eea10a628e497987d7685b2e6718052025-08-20T03:10:32ZengNature PortfolioCommunications Biology2399-36422025-05-018111210.1038/s42003-025-08187-5Comparative analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous taggingChiharu Tei0Shoji Hata1Akira Mabuchi2Shotaro Okuda3Kei K. Ito4Mariya Genova5Masamitsu Fukuyama6Shohei Yamamoto7Takumi Chinen8Atsushi Toyoda9Daiju Kitagawa10Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, BunkyoDepartment of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, BunkyoDepartment of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, BunkyoDepartment of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, BunkyoDepartment of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, BunkyoZentrum für Molekulare Biologie, Universität Heidelberg, DKFZ-ZMBH AllianzDepartment of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, BunkyoDepartment of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, BunkyoDepartment of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, BunkyoComparative Genomics Laboratory and Advanced Genomics Center, National Institute of Genetics, MishimaDepartment of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, BunkyoAbstract CRISPR-mediated endogenous tagging is a powerful tool in biological research. Inhibiting the non-homologous end joining (NHEJ) pathway has been shown to improve the low efficiency of accurate knock-in via homology-directed repair (HDR). However, the influence of alternative double-stranded break (DSB) repair pathways on knock-in remains to be fully explored. In this study, our long-read amplicon sequencing analysis reveals various patterns of imprecise repair in CRISPR-mediated knock-in, even with NHEJ inhibition. Further suppressing either microhomology-mediated end joining (MMEJ) or single-strand annealing (SSA) reduces nucleotide deletions around the cut site, thereby elevating knock-in accuracy. Additionally, imprecise donor integration is reduced by inhibiting SSA, but not MMEJ. Particularly, SSA suppression reduced asymmetric HDR, a specific imprecise integration pattern, which we further confirm using a novel reporter system. These findings demonstrate the complex interplay of multiple DSB repair pathways in CRISPR-mediated knock-in and offer novel strategies, including SSA pathway targeting, to improve precise gene editing efficiency.https://doi.org/10.1038/s42003-025-08187-5 |
| spellingShingle | Chiharu Tei Shoji Hata Akira Mabuchi Shotaro Okuda Kei K. Ito Mariya Genova Masamitsu Fukuyama Shohei Yamamoto Takumi Chinen Atsushi Toyoda Daiju Kitagawa Comparative analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging Communications Biology |
| title | Comparative analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging |
| title_full | Comparative analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging |
| title_fullStr | Comparative analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging |
| title_full_unstemmed | Comparative analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging |
| title_short | Comparative analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging |
| title_sort | comparative analysis of multiple dna double strand break repair pathways in crispr mediated endogenous tagging |
| url | https://doi.org/10.1038/s42003-025-08187-5 |
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