Abstract
Xenopus laevis have proven to be a useful system for rapid generation and analysis of transgenic models of human retinal disease. However, experimental approaches in this system were limited by lack of a robust knockdown or knockout technology. Here we describe a protocol for generation of Cas9-edited X. laevis embryos. The technique introduces point mutations into the genome of X. laevis resulting in in-frame and out-of-frame insertions and deletions that allow modeling of both dominant and recessive human diseases and efficiently generates gene knockdown and knockout. Our techniques can produce high-frequency gene editing in X. laevis, permitting analysis in the F0 generation.
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Acknowledgments
This research was supported by the Natural Sciences and Engineering Research Council of Canada and the Foundation Fighting Blindness (Canada). B630N antibody was a gift of Dr. W. Clay Smith.
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Feehan, J.M., Stanar, P., Tam, B.M., Chiu, C., Moritz, O.L. (2019). Generation and Analysis of Xenopus laevis Models of Retinal Degeneration Using CRISPR/Cas9. In: Weber, B.H.F., Langmann, T. (eds) Retinal Degeneration. Methods in Molecular Biology, vol 1834. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8669-9_14
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DOI: https://doi.org/10.1007/978-1-4939-8669-9_14
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-8668-2
Online ISBN: 978-1-4939-8669-9
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