- Letter to the Editor
- Open Access
Targeted gene disruption in Xenopus laevis using CRISPR/Cas9
© Wang et al.; licensee BioMed Central. 2015
Received: 23 February 2015
Accepted: 25 March 2015
Published: 14 April 2015
To test if the CRISPR/Cas9 system can mediate targeted gene disruption in Xenopus laevis, we targeted ptf1a/p48 and tyrosinase in this species and found that in addition to high sequence disruption efficiency, clear phenotypes were observed in G0 embryos. ptf1a/p48-targeted X. laevis embryos can mimic Xenopus tropicalis ptf1a/p48-null mutant tadpoles with respect to the loss of pdip expression. Simultaneous disruption of two X. laevis tyrosinase homeologs leads to almost full albinism. Our data indicate that CRISPR/Cas9 system is a simple and efficient tool for targeted gene disruption in X. laevis. It can be used for phenotype analysis in G0 embryos.
Since its introduction to modern biology in the 1950s, X. laevis has played a central role in most disciplines of biomedical research including developmental biology, biochemistry, and molecular biology . Its allotetraploid genome represents a common polyploidy in amphibians, which challenges genetic studies on this species . Fortunately, by the mid 1990s, X. tropicalis, the only diploid species in the Xenopus genus, was adopted as a genetically tractable complement to the classic model X. laevis. Together, the two frog species provide a unique and powerful system for addressing genome duplication/evolution, functional genomics, and human disease modelling at the post-genomic era .
Gene knockdown in X. laevis has been largely dependent on antisense Morpholino oligomers since 2000, which suffers from some off-target effects . A recent systematic analysis reveals poor phenotypic correlation between published Morpholino-induced morphants and mutant lines in zebrafish , further highlighting the off-target effects of Morpholino oligomers. Transcription activator-like effector nuclease (TALEN) was shown effective for targeted gene disruption in X. laevis G0 embryos [1,5-7]. With the establishment of efficient targeted gene disruption in X. tropicalis using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR/Cas9) system , it is worth addressing if this simple method applies to X. laevis.
CRISPR/Cas9 mediated disruption of ptf1a/p48 in X. laevis G0 embryos phenocopies X. tropicalis ptf1a/p48 mutants
Simultaneous disruption of two X. laevis tyrosinase homeologs leads to albinism
Due to Morpholino’s potential off-target effects, a recent study recommends mutant phenotypes as the standard metric to define gene function in zebrafish . Our previous study did not detect any CRISPR/Cas9-induced off-target effects in X. tropicalis . The issue of CRISPR/Cas9’s off-target effects in X. laevis remains to be defined. Nevertheless, our data demonstrate that CRISPR/Cas9 is equally a superb tool for targeted gene disruption in X. laevis as in X. tropicalis. It can be used for an immediate phenotype evaluation in X. laevis G0 embryos. It should be noted that the allotetraploid genome and longer generation time (1–2 years) of X. laevis makes it impractical to do genetic research. For establishment of stable knockout lines, we recommend to use the diploid frog X. tropicalis that has much shorter generation time (4–6 months).
This work was supported in part by National Basic Research Program of China (2015CB942803), the National Natural Science Foundation of China (31471367, 31271554, and 31301192), Shenzhen Key Laboratory of Cell Microenvironment (ZDSYS20140509142721429), and the Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. This research was also supported in part by the Intramural Research Program of NICHD, NIH.
- Schmitt SM, Gull M, Brändli AW. Engineering Xenopus embryos for phenotypic drug discovery screening. Adv Drug Deliv Rev. 2014;69:225–46.View ArticlePubMedGoogle Scholar
- Tinsley RC, Kobel HR. Allopolyploid speciation. In: Tinsley RC, Kobel HR, editors. The Biology of Xenopus. Oxford: Clarendon Press; 1996. p. 391–402.Google Scholar
- Eisen JS, Smith JC. Controlling morpholino experiments: don’t stop making antisense. Development. 2008;135:1735–43.View ArticlePubMedGoogle Scholar
- Kok FO, Shin M, Ni CW, Gupta A, Grosse AS, van Impel A, et al. Reverse genetic screening reveals poor correlation between Morpholino-Induced and mutant phenotypes in Zebrafish. Dev Cell. 2015;32:97–108.View ArticlePubMedGoogle Scholar
- Lei Y, Guo X, Deng Y, Chen Y, Zhao H. Generation of gene disruptions by transcription activator-like effector nucleases (TALENs) in Xenopus tropicalis embryos. Cell Biosci. 2013;3:21.View ArticlePubMed CentralPubMedGoogle Scholar
- Suzuki KT, Isoyama Y, Kashiwagi K, Sakuma T, Ochiai H, Sakamoto N, et al. High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos. Biol Open. 2013;2:448–52.View ArticlePubMed CentralPubMedGoogle Scholar
- Sakane Y, Sakuma T, Kashiwagi K, Kashiwagi A, Yamamoto T, Suzuki KT. Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator‐like effector nucleases. Dev Growth Differ. 2014;56:108–14.View ArticlePubMedGoogle Scholar
- Guo X, Zhang T, Hu Z, Zhang Y, Shi Z, Wang Q, et al. Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis. Development. 2014;141:707–14.View ArticlePubMedGoogle Scholar
- Krapp A, Knöfler M, Ledermann B, Bürki K, Berney C, Zoerkler N, et al. The bHLH protein PTF1-p48 is essential for the formation of the exocrine and the correct spatial organization of the endocrine pancreas. Genes Dev. 1998;12:3752–63.View ArticlePubMed CentralPubMedGoogle Scholar
- Afelik S, Chen Y, Pieler T. Combined ectopic expression of Pdx1 and Ptf1a/p48 results in the stable conversion of posterior endoderm into endocrine and exocrine pancreatic tissue. Genes Dev. 2006;20:1441–6.View ArticlePubMed CentralPubMedGoogle Scholar
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