Genotyping of CRISPR/Cas9 Genome Edited Xenopus tropicalis.

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 Furthermore, we provide a previously unpublished pipe-line for rapid genotyping of F1 offspring-high resolution melting analysis (HRMA) and sequence trace decomposition-procured from breeding with F0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.