Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes
The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, e...
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| Language: | English |
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Taylor & Francis Group
2015-10-01
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| Series: | BioTechniques |
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| Online Access: | https://www.future-science.com/doi/10.2144/000114339 |
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| author | Bangmei Wang Kunyu Li Amy Wang Michelle Reiser Thom Saunders Richard F Lockey Jia-Wang Wang |
| author_facet | Bangmei Wang Kunyu Li Amy Wang Michelle Reiser Thom Saunders Richard F Lockey Jia-Wang Wang |
| author_sort | Bangmei Wang |
| collection | DOAJ |
| description | The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general. |
| format | Article |
| id | doaj-art-9a1163b106d945cda978afdc09fb32ff |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2015-10-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-9a1163b106d945cda978afdc09fb32ff2025-08-20T02:25:55ZengTaylor & Francis GroupBioTechniques0736-62051940-98182015-10-0159420120810.2144/000114339Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotesBangmei Wang0Kunyu Li1Amy Wang2Michelle Reiser3Thom Saunders4Richard F Lockey5Jia-Wang Wang61Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL1Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL1Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL1Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL2University of Michigan, Ann Arbor, MI1Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL1Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FLThe clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.https://www.future-science.com/doi/10.2144/000114339CRISPR cleavagedouble-strand break (DSB)homology-directed-repair (HDR)embryonic stem (ES) cellszygote |
| spellingShingle | Bangmei Wang Kunyu Li Amy Wang Michelle Reiser Thom Saunders Richard F Lockey Jia-Wang Wang Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes BioTechniques CRISPR cleavage double-strand break (DSB) homology-directed-repair (HDR) embryonic stem (ES) cells zygote |
| title | Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes |
| title_full | Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes |
| title_fullStr | Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes |
| title_full_unstemmed | Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes |
| title_short | Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes |
| title_sort | highly efficient crispr hdr mediated knock in for mouse embryonic stem cells and zygotes |
| topic | CRISPR cleavage double-strand break (DSB) homology-directed-repair (HDR) embryonic stem (ES) cells zygote |
| url | https://www.future-science.com/doi/10.2144/000114339 |
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