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

References to SciGraph publications

  • 2018-01. Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs in NATURE BIOMEDICAL ENGINEERING
  • 2017-12. CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing in NATURE COMMUNICATIONS
  • 2014-12. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9 in NATURE COMMUNICATIONS
  • 2009. APC and Its Modifiers in Colon Cancer in APC PROTEINS
  • 2016-12. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR in GENOME BIOLOGY
  • 2016-12. CRISPR/Cas9 mediated knockout of rb1 and rbl1 leads to rapid and penetrant retinoblastoma development in Xenopus tropicalis in SCIENTIFIC REPORTS
  • 2014-12. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation in NATURE BIOTECHNOLOGY
  • 2015-10. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo in NATURE METHODS
  • 2017-12. Modeling Dominant and Recessive Forms of Retinitis Pigmentosa by Editing Three Rhodopsin-Encoding Genes in Xenopus Laevis Using Crispr/Cas9 in SCIENTIFIC REPORTS
  • 2017-12. CRISPR/Cas9-mediated genome editing induces exon skipping by alternative splicing or exon deletion in GENOME BIOLOGY
  • 2017-06. An Interspecies Heart-to-Heart: Using Xenopus to Uncover the Genetic Basis of Congenital Heart Disease in CURRENT PATHOBIOLOGY REPORTS
  • 2016-12. Efficient genome editing of genes involved in neural crest development using the CRISPR/Cas9 system in Xenopus embryos in CELL & BIOSCIENCE
  • 2015-06. Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains in NATURE BIOTECHNOLOGY
  • 2016-02. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 in NATURE BIOTECHNOLOGY
  • Book

    TITLE

    Xenopus

    ISBN

    978-1-4939-8783-2
    978-1-4939-8784-9

    Author Affiliations

    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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