The DOI matches its Crossref record; 60 of the 60 references it cites were found in a registry or an index.
Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins
Open accessAbstract
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 ( E fficient a dditions with s sDNA i nserts-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.
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Self-citationNuclear receptor REVERBα is a state-dependent regulator of liver energy metabolism
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Metadata
- Authors
- Quadros, R. M., Miura, H., Harms, D. W., Akatsuka, H., Sato, T., Aida, T., Redder, R., Richardson, G. P., Inagaki, Y., Sakai, D., Buckley, S. M., Seshacharyulu, P., Batra, S. K., Behlke, M. A., Zeiner, S. A., Jacobi, A. M., Izu, Y., Thoreson, W. B., Urness, L. D., Mansour, S. L., Ohtsuka, M., & Gurumurthy, C. B.
- Venue
- Genome Biology
- Published
- 2017-05-17
- Volume
- 18
- Issue
- 1
- Pages
- 92-92
- ISSN
- 1465-6906 (ISSN-L)
- 1474-760X
- OpenAlex
- W2614850398
- Cited by
- 513
- References
- 60
- FWCI
- 16.01
- Type
- journal article
- Language
- en
- Open access
- gold
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- Quadros, R. M., Miura, H., Harms, D. W., Akatsuka, H., Sato, T., Aida, T., Redder, R., Richardson, G. P., Inagaki, Y., Sakai, D., Buckley, S. M., Seshacharyulu, P., Batra, S. K., Behlke, M. A., Zeiner, S. A., Jacobi, A. M., Izu, Y., Thoreson, W. B., Urness, L. D., . . . Gurumurthy, C. B. (2017). 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. https://doi.org/10.1186/s13059-017-1220-4
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Bibliographic data: Semantic Scholar, OpenAlex, Crossref.
Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).
Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.
