Chapter title |
CRISPR-Cas9-Based Functional Analysis in Amphibians: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl.
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Chapter number | 26 |
Book title |
Genome Editing in Animals
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Published in |
Methods in molecular biology, January 2023
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DOI | 10.1007/978-1-0716-3016-7_26 |
Pubmed ID | |
Book ISBNs |
978-1-07-163015-0, 978-1-07-163016-7
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Authors |
Suzuki, Miyuki, Iida, Midori, Hayashi, Toshinori, Suzuki, Ken-Ichi T, Suzuki, Ken-ichi T. |
Abstract |
Amphibians have made many fundamental contributions to our knowledge, from basic biology to biomedical research on human diseases. Current genome editing tools based on the CRISPR-Cas system enable us to perform gene functional analysis in vivo, even in non-model organisms. We introduce here a highly efficient and easy protocol for gene knockout, which can be used in three different amphibians seamlessly: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl. As it utilizes Cas9 ribonucleoprotein complex (RNP) for injection, this cloning-free method enables researchers to obtain founder embryos with a nearly complete knockout phenotype within a week. To evaluate somatic mutation rate and its correlation to the phenotype of a Cas9 RNP-injected embryo (crispant), we also present accurate and cost-effective genotyping methods using pooled amplicon-sequencing and a user-friendly web-based tool. |
X Demographics
Geographical breakdown
Country | Count | As % |
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Germany | 1 | 33% |
Unknown | 2 | 67% |
Demographic breakdown
Type | Count | As % |
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Members of the public | 3 | 100% |