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

Gandhi, Shashank and Li, Yuwei and Tang, Weiyi and Christensen, Jens B. and Urrutia, Hugo A. and Vieceli, Felipe M. and Piacentino, Michael L. and Bronner, Marianne E. (2021) A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos. Development, 148 (7). Art. No. dev193565. ISSN 0950-1991. PMCID PMC8077534. doi:10.1242/dev.193565.

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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.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle
Gandhi, Shashank0000-0002-4081-4338
Li, Yuwei0000-0001-7753-4869
Tang, Weiyi0000-0002-1279-1001
Christensen, Jens B.0000-0003-4617-3866
Urrutia, Hugo A.0000-0002-2970-6918
Vieceli, Felipe M.0000-0001-5142-8224
Piacentino, Michael L.0000-0003-1773-031X
Bronner, Marianne E.0000-0003-4274-1862
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 (, 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.
Funding AgencyGrant Number
American Heart Association18PRE34050063
Otto Brunns Fund82318917
Subject Keywords:CRISPR-Cas9, Chick embryology, Clonal analysis, Retroviruses, Migration, Genome editing, Neural crest
Issue or Number:7
PubMed Central ID:PMC8077534
Record Number:CaltechAUTHORS:20210311-131531358
Persistent URL:
Official Citation:Shashank Gandhi, Yuwei Li, Weiyi Tang, Jens B. Christensen, Hugo A. Urrutia, Felipe M. Vieceli, Michael L. Piacentino, Marianne E. Bronner; A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos. Development 1 April 2021; 148 (7): dev193565. doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108401
Deposited By: Tony Diaz
Deposited On:12 Mar 2021 17:49
Last Modified:05 Aug 2021 16:37

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