In this chapter, we convey a state-of-the art update to the 2014 Nakayama protocol for CRISPR/Cas9 genome engineering in Xenopus tropicalis (X. tropicalis). We discuss in depth, gRNA design software and rules, gRNA synthesis, and procedures for tissue- and tissue-specific CRISPR/Cas9 genome editing by targeted microinjection in X. tropicalis embryos. We demonstrate the methodology by which any standard equipped Xenopus researcher with microinjection experience can generate F0 CRISPR/Cas9 mediated mosaic mutants (crispants) within one to two work-week(s). The described methodology allows CRISPR/Cas9 efficiencies to be high enough to read out phenotypic consequences, and thus perform gene function analysis, in the F0 crispant. Additionally, we provide the framework for performing multiplex tissue-specific CRISPR/Cas9 experiments generating crispants mosaic mutant in up to four genes simultaneously, which can be of importance for Laevis researchers aiming to target by CRISPR/Cas9 both the S and L homeolog of a gene simultaneously. Finally, we discuss off-target concerns, how to minimize these and ways to rapidly bypass reviewer off-target critique by exploiting the advantages of X. tropicalis.
Keywords: CRISPR/Cas9; Disease model; Multiplex; Tissue-specific; Xenopus tropicalis.