One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering
Open accessAbstract
The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.
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The desired recombination efficacy of HDR often occurs infrequently, which presents enormous challenges for robust applications.
“However, the desired recombination efficacy often occurs infrequently, which presents enormous challenges for robust applications [61, 82–84].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [84]
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- Authors
- Yang, H., Wang, H., Shivalila, C. S., Cheng, A. W., Shi, L., & Jaenisch, R.
- Affiliations
- Whitehead Institute for Biomedical Research (US)
- Corresponding author
- Jaenisch, Rudolf
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2013-09
- Volume
- 154
- Issue
- 6
- Pages
- 1370-1379
- ISSN
- 0092-8674 (ISSN-L)
- PMC
- PMC3961003
- OpenAlex
- W2140754284
- Cited by
- 1,596
- References
- 29
- Citations by year
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- FWCI
- 76.80
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Cell (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-nd)
- PMC
- Funders
- National Institutes of Health
- National Cancer Institute (R37CA084198)
- Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD045022, R37HD045022)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Keywords
- conditional mutant mice, reporter mice, zygote injection, MeCP2, Nanog, SOX2, Oct4, off-target mutations, embryonic stem cells, guide RNA
- MeSH terms
- Animals, Base Sequence, Genetic Engineering, Mutation, Gene Targeting, Mice
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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