Abstract
Using TALEN or CRISPR/Cas system to induce small indels into coding sequences has been implicated in broad applications for reverse genetic studies of many organisms including zebrafish. However, complete deletion of a large gene or noncoding gene(s) or removing a large genomic fragment spanning several genes or other chromosomal elements is preferred in various cases, as well as inducing chromosomal inversions. Here, we describe the detailed protocols for the generation of chromosomal deletion mutations mediated by Cas9 and a pair of gRNAs and the evaluation for the efficiencies in F0 founder fish and of germline transmission.
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Acknowledgment
We thank Zhanxiang Wang, Da Liu, and other members in our lab for their efforts on optimizing the CRISPR/Cas applications in zebrafish. This work was partially supported by the National Natural Science Foundation of China (31110103904, 81371264), the 973 Program of the Ministry of Science and Technology of China (2012CB945101, 2015CB942803), and the Seeding Grant for Medicine and Life Sciences of Peking University (2014-MB-06).
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Xiao, A., Zhang, B. (2016). Generation of Targeted Genomic Deletions Through CRISPR/Cas System in Zebrafish. In: Kawakami, K., Patton, E., Orger, M. (eds) Zebrafish. Methods in Molecular Biology, vol 1451. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3771-4_5
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DOI: https://doi.org/10.1007/978-1-4939-3771-4_5
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-3771-4
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