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 (RIA) 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, neural tube and segmental plate in vivo, respectively. Together, this approach enables knocking out genes of interest in identifiable cells in living embryos and can be broadly applied to numerous genes in different embryonic tissues.

Schwann cell precursors represent a neural crest‐like state with biased multipotency

“…To test the function of the “hub”‐specific transcription factor Sox8 , we electroporated the trunk neural tube of HH11 chick embryos with a single plasmid containing gRNA, Cas9 and a citrine reporter (Gandhi et al , 2021) to knockout Sox8 in neural crest‐derived cells (Fig 4A and B). Embryos were then allowed to develop until HH24‐25 (Fig 4C), by which time the dorsal root ganglia (DRG) and visceral nerves had developed (Fig 4H).…”

“…Crispr constructs were designed and made according to the protocol by Gandhi et al (2021). The plasmid contains a sequence for Cas9, citrine and a guide RNA.…”