Genome engineering tools for avian embryos
Updated: Jun 19, 2021
During the first year of my Ph.D. in Dr. Marianne Bronner's lab at the California Institute of Technology, I optimized the CRISPR-Cas9 system for genome editing in gastrula- and neurula-stage chicken embryos (Gandhi et al., Developmental Biology, 2017). This improved approach has enabled the field of avian developmental biology to interrogate epistatic relationships between key neural crest genes during neural crest induction. The reagents are available through the Bronner lab's webpage on Addgene.
Image: Reagents encoding Cas9 and gene-specific guide RNAs can be electroporated in chick gastrula-stage embryos. The efficiency and extent of knockout can then be assayed by antibody staining (left and middle panels) or in situ hybridization (right panel).
I have since further improved upon the CRISPR toolbox and devised a combinatorial approach for lineage analysis and CRISPR-Cas9-mediated genome editing that employs replication-incompetent avian retroviruses to study experimentally perturbed cells in an otherwise normal environment in several tissues of the chick embryo. To achieve this, I have engineered an all-in-one plasmid that encodes Cas9 protein, gene-specific guide RNA (gRNA), and a fluorescent marker within the same construct (Gandhi et al., Development, 2021). Using transfection- and electroporation-based approaches, we have shown that this strategy can be implemented to perturb gene function in early embryos as well as human cell lines. This work finally makes it possible to challenge a cell’s developmental potential and interrogate the role of particular genes in developmental processes by enabling their knockout in permanently-labeled cells in living embryos.
Movie: Unperturbed neural crest cells (labeled in green) delaminate and migrate away from the developing neural tube.
Movie: Following knockout of the neural crest gene Sox10 using the single-plasmid approach, neural crest cells fail to migrate properly and undergo premature apoptosis.