Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins
Abstract Background Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homo...
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| Format: | Article |
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BMC
2017-05-01
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| Series: | Genome Biology |
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| Online Access: | http://link.springer.com/article/10.1186/s13059-017-1220-4 |
| _version_ | 1819212795643691008 |
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| author | Rolen M. Quadros Hiromi Miura Donald W. Harms Hisako Akatsuka Takehito Sato Tomomi Aida Ronald Redder Guy P. Richardson Yutaka Inagaki Daisuke Sakai Shannon M. Buckley Parthasarathy Seshacharyulu Surinder K. Batra Mark A. Behlke Sarah A. Zeiner Ashley M. Jacobi Yayoi Izu Wallace B. Thoreson Lisa D. Urness Suzanne L. Mansour Masato Ohtsuka Channabasavaiah B. Gurumurthy |
| author_facet | Rolen M. Quadros Hiromi Miura Donald W. Harms Hisako Akatsuka Takehito Sato Tomomi Aida Ronald Redder Guy P. Richardson Yutaka Inagaki Daisuke Sakai Shannon M. Buckley Parthasarathy Seshacharyulu Surinder K. Batra Mark A. Behlke Sarah A. Zeiner Ashley M. Jacobi Yayoi Izu Wallace B. Thoreson Lisa D. Urness Suzanne L. Mansour Masato Ohtsuka Channabasavaiah B. Gurumurthy |
| author_sort | Rolen M. Quadros |
| collection | DOAJ |
| description | Abstract Background Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient. Results Here we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live offspring. Conclusions Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources. |
| first_indexed | 2024-12-23T06:48:39Z |
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| id | doaj.art-cc09dd43b09b4403b9ad7415943adf1e |
| institution | Directory Open Access Journal |
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| language | English |
| last_indexed | 2024-12-23T06:48:39Z |
| publishDate | 2017-05-01 |
| publisher | BMC |
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| series | Genome Biology |
| spelling | doaj.art-cc09dd43b09b4403b9ad7415943adf1e2022-12-21T17:56:29ZengBMCGenome Biology1474-760X2017-05-0118111510.1186/s13059-017-1220-4Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteinsRolen M. Quadros0Hiromi Miura1Donald W. Harms2Hisako Akatsuka3Takehito Sato4Tomomi Aida5Ronald Redder6Guy P. Richardson7Yutaka Inagaki8Daisuke Sakai9Shannon M. Buckley10Parthasarathy Seshacharyulu11Surinder K. Batra12Mark A. Behlke13Sarah A. Zeiner14Ashley M. Jacobi15Yayoi Izu16Wallace B. Thoreson17Lisa D. Urness18Suzanne L. Mansour19Masato Ohtsuka20Channabasavaiah B. Gurumurthy21Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical CenterDepartment of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of MedicineMouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical CenterDepartment of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of MedicineDepartment of Host Defense Mechanism, Division of Basic Medical Science and Molecular Medicine, Tokai University School of MedicineLaboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU)High-Throughput DNA Sequencing and Genotyping Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical CenterSussex Neuroscience, University of SussexCenter for Matrix Biology and Medicine, Graduate School of Medicine, Tokai UniversityThe Institute of Medical Sciences, Tokai UniversityDepartment of Genetics, Cell Biology & Anatomy, College of Medicine, University of Nebraska Medical CenterDepartment of Biochemistry and Molecular Biology, University of Nebraska Medical CenterDepartment of Biochemistry and Molecular Biology, University of Nebraska Medical CenterIntegrated DNA Technologies, Inc.Integrated DNA Technologies, Inc.Integrated DNA Technologies, Inc.Department of Animal Risk Management, Chiba Institute of ScienceTruhlsen Eye Institute and Department of Ophthalmology & Visual Sciences, University of Nebraska Medical CenterDepartment of Human Genetics, University of UtahDepartment of Human Genetics, University of UtahDepartment of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of MedicineMouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical CenterAbstract Background Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient. Results Here we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live offspring. Conclusions Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.http://link.springer.com/article/10.1186/s13059-017-1220-4CRISPR/Cas9Homology directed repairEasi-CRISPRlong ssDNA donorsCRISPR ribonucleoproteinsCre-LoxP |
| spellingShingle | Rolen M. Quadros Hiromi Miura Donald W. Harms Hisako Akatsuka Takehito Sato Tomomi Aida Ronald Redder Guy P. Richardson Yutaka Inagaki Daisuke Sakai Shannon M. Buckley Parthasarathy Seshacharyulu Surinder K. Batra Mark A. Behlke Sarah A. Zeiner Ashley M. Jacobi Yayoi Izu Wallace B. Thoreson Lisa D. Urness Suzanne L. Mansour Masato Ohtsuka Channabasavaiah B. Gurumurthy Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins Genome Biology CRISPR/Cas9 Homology directed repair Easi-CRISPR long ssDNA donors CRISPR ribonucleoproteins Cre-LoxP |
| title | Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins |
| title_full | Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins |
| title_fullStr | Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins |
| title_full_unstemmed | Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins |
| title_short | Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins |
| title_sort | easi crispr a robust method for one step generation of mice carrying conditional and insertion alleles using long ssdna donors and crispr ribonucleoproteins |
| topic | CRISPR/Cas9 Homology directed repair Easi-CRISPR long ssDNA donors CRISPR ribonucleoproteins Cre-LoxP |
| url | http://link.springer.com/article/10.1186/s13059-017-1220-4 |
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