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Published April 2021 | Supplemental Material + Published
Journal Article Open

A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos

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.

Additional Information

© 2021. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. Received 3 June 2020; Accepted 23 February 2021. For technical assistance, we thank Andres Collazo, Steven Wilbert and Giada Spigolon with the Caltech Biological Imaging facility of the Beckman Institute. We thank members of the Bronner lab for helpful discussions. Author contributions: Conceptualization: S.G., M.E.B.; Methodology: S.G., Y.L.; Validation: S.G.; Formal analysis: S.G.; Investigation: S.G., Y.L., W.T., J.B.C., H.A.U., F.M.V., M.L.P., M.E.B.; Resources: Y.L.; Writing - original draft: S.G., Y.L., W.T., H.A.U., M.E.B.; Writing - review & editing: S.G., Y.L., M.L.P., M.E.B.; Visualization: S.G., W.T., M.L.P.; Supervision: S.G., M.E.B.; Funding acquisition: M.E.B. This work is supported by the National Institutes of Health (R01DE027568 to M.E.B. and K99DE029240 to M.L.P.), the American Heart Association (predoctoral fellowship 18PRE34050063 to S.G.) and the Otto Brunns Fund 82318917 to J.B.C. Open access funding provided by the California Institute of Technology (CALTECH). Deposited in PMC for immediate release. The authors declare no competing or financial interests.

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Additional details

Created:
August 20, 2023
Modified:
December 22, 2023