1. A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos.
- Author
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Gandhi S, Li Y, Tang W, Christensen JB, Urrutia HA, Vieceli FM, Piacentino ML, and Bronner ME
- Subjects
- Animals, Chick Embryo, Gene Expression Regulation, Developmental, Gene Knockout Techniques methods, Neural Crest metabolism, PAX6 Transcription Factor genetics, PAX7 Transcription Factor, RNA, Guide, CRISPR-Cas Systems genetics, SOXE Transcription Factors genetics, CRISPR-Cas Systems, Gene Editing methods, Plasmids genetics
- Abstract
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 β-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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)
- Published
- 2021
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