Abstract
The targeted nuclease revolution (ZFN, TALEN, and CRISPR/Cas9) has led to a myriad of reports describing genotyping methodologies for genome edited founders (F0—crispants) and their offspring (F1). As such, choosing a specific genotyping methodology for your Xenopus CRISPR/Cas9 experiments can be challenging. In this chapter we will discuss, with emphasis on Xenopus tropicalis (X. tropicalis), different methods for assessing genome editing efficiencies within F0 CRISPR/Cas9 founders and for identification of their hetero-, compound hetero-, and homozygous mutant F1 offspring. For F0 crispants, we will provide the protocols and the respective (dis)advantages of genotyping with heteroduplex mobility assay (HMA), subclone Sanger sequencing, and sequence trace decomposition. Furthermore, we provide a previously unpublished pipe-line for rapid genotyping of F1 offspring—high resolution melting analysis (HRMA) and sequence trace decomposition—procured from breeding with F0 crispants. As such, we report here the current state-of-the-art cost- and time-effective approaches to perform genotyping of CRISPR/Cas9 experiments for the Xenopus tropicalis researcher.
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Acknowledgments
The authors would like to thank Dr. Tom Van Nieuwenhuysen for the initial establishment of the HMA assay and the subcloning/Sanger sequencing methodologies within the research unit. Furthermore, the authors would like to thank Lana Hellebaut for critical proofreading of this chapter. Research in the authors’ laboratory is supported by the Research Foundation – Flanders (FWO-Vlaanderen) (grants G0A1515N and G029413 N), by the Belgian Science Policy (Interuniversity Attraction Poles - IAP7/07) and by the Concerted Research Actions from Ghent University (BOF15/GOA/011). Further support was obtained by the Hercules Foundation, Flanders (grant AUGE/11/14) and the Desmoid Tumor Research Foundation.
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Naert, T., Vleminckx, K. (2018). Genotyping of CRISPR/Cas9 Genome Edited Xenopus tropicalis. In: Vleminckx, K. (eds) Xenopus. Methods in Molecular Biology, vol 1865. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8784-9_5
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DOI: https://doi.org/10.1007/978-1-4939-8784-9_5
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