ArticleCell & bioscience2016
Efficient genome editing of genes involved in neural crest development using the CRISPR/Cas9 system in Xenopus embryos.
Article in Cell & bioscience, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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Who cites it
5 citing papers in PubMed, 13 citations in OpenAlex.
- Rs1hGene therapy · 2022Article
- Genetic resiliency associated with dominant lethalCell reports. Medicine · 2022Article
- Advancing genetic and genomic technologies deepen the pool for discovery in Xenopus tropicalis.Developmental dynamics : an official publication of the American Association of Anatomists · 2019Review
- MAPK and PI3K signaling: At the crossroads of neural crest development.Developmental biology · 2018Review
- Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus.Journal of visualized experiments : JoVE · 2018Article
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Authors and funding
14 authors at 4 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundThe RNA guided CRISPR/Cas9 nucleases have been proven to be effective for gene disruption in various animal models including Xenopus tropicalis. The neural crest (NC) is a transient cell population during embryonic development and contributes to a large variety of tissues. Currently, loss-of-function studies on NC development in X. tropicalis are largely based on morpholino antisense oligonucleotide. It is worthwhile establishing targeted gene knockout X. tropicails line using CRISPR/Cas9 system to study NC development.
methodsWe utilized CRISPR/Cas9 to disrupt genes that are involved in NC formation in X. tropicalis embryos. A single sgRNA and Cas9 mRNA synthesized in vitro, were co-injected into X. tropicalis embryos at one-cell stage to induce single gene disruption. We also induced duplex mutations, large segmental deletions and inversions in X. tropicalis by injecting Cas9 and a pair of sgRNAs. The specificity of CRISPR/Cas9 was assessed in X. tropicalis embryos and the Cas9 nickase was used to reduce the off-target cleavages. Finally, we crossed the G0 mosaic frogs with targeted mutations to wild type frogs and obtained the germline transmission.
resultsTotal 16 target sites in 15 genes were targeted by CRISPR/Cas9 and resulted in successful indel mutations at 14 loci with disruption efficiencies in a range from 9.3 to 57.8 %. Furthermore, we demonstrated the feasibility of generation of duplex mutations, large segmental deletions and inversions by using Cas9 and a pair of sgRNAs. We observed that CRISPR/Cas9 displays obvious off-target effects at some loci in X. tropicalis embryos. Such off-target cleavages was reduced by using the D10A Cas9 nickase. Finally, the Cas9 induced indel mutations were efficiently passed to G1 offspring.
conclusionOur study proved that CRISPR/Cas9 could mediate targeted gene mutation in X. tropicalis with high efficiency. This study expands the application of CRISPR/Cas9 platform in X. tropicalis and set a basis for studying NC development using genetic approach.
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