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A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos.
Gandhi, Shashank; Li, Yuwei; Tang, Weiyi; Christensen, Jens B; Urrutia, Hugo A; Vieceli, Felipe M; Piacentino, Michael L; Bronner, Marianne E.
Afiliação
  • Gandhi S; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
  • Li Y; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
  • Tang W; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
  • Christensen JB; Department of Neuroscience, University of Copenhagen, Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark.
  • Urrutia HA; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
  • Vieceli FM; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
  • Piacentino ML; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
  • Bronner ME; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Development ; 148(7)2021 04 01.
Article em En | MEDLINE | ID: mdl-33688075
An important strategy for establishing mechanisms of gene function during development is through mutation of individual genes and analysis of subsequent effects on cell behavior. Here, we present a single-plasmid approach for genome editing in chick embryos to study experimentally perturbed cells in an otherwise normal embryonic environment. To achieve this, we have engineered a plasmid that encodes Cas9 protein, gene-specific guide RNA (gRNA), and a fluorescent marker within the same construct. Using transfection- and electroporation-based approaches, we show that this construct can be used to perturb gene function in early embryos as well as human cell lines. Importantly, insertion of this cistronic construct into replication-incompetent avian retroviruses allowed us to couple gene knockouts with long-term lineage analysis. We demonstrate the application of our newly engineered constructs and viruses by perturbing ß-catenin in vitro and Sox10, Pax6 and Pax7 in the neural crest, retina, and neural tube and segmental plate in vivo, respectively. Together, this approach enables genes of interest to be knocked out in identifiable cells in living embryos and can be broadly applied to numerous genes in different embryonic tissues.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Plasmídeos / Sistemas CRISPR-Cas / Edição de Genes Tipo de estudo: Prognostic_studies Limite: Animals Idioma: En Ano de publicação: 2021 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Plasmídeos / Sistemas CRISPR-Cas / Edição de Genes Tipo de estudo: Prognostic_studies Limite: Animals Idioma: En Ano de publicação: 2021 Tipo de documento: Article