Methods for CRISPR/Cas9 Xenopus tropicalis Tissue-Specific Multiplex Genome Engineering View Full Text


Ontology type: schema:Chapter     


Chapter Info

DATE

2018-08-28

AUTHORS

Thomas Naert , Kris Vleminckx

ABSTRACT

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. More... »

PAGES

33-54

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/978-1-4939-8784-9_3

DOI

http://dx.doi.org/10.1007/978-1-4939-8784-9_3

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1106364916

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/30151757


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