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

Shashank Gandhi,Marianne E Bronner,Jens B Christensen,Weiyi Tang,Michael L Piacentino,Felipe M Vieceli,Yuwei Li,Hugo A Urrutia

doi:10.1242/dev.193565

Shashank Gandhi, Marianne E Bronner + Show 6 more

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https://doi.org/10.1242/dev.193565

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Abstract

ABSTRACTAn 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.

Highlights

Advent of CRISPR-Cas9 is a highlight of the past decade that has enabled genome editing across multiple species (Chen et al, 2013; Cong et al, 2013; Dickinson et al, 2013; Gagnon et al, 2014; Stolfi et al, 2014)
Because Cas9 and guide RNA (gRNA) were delivered via separate constructs, a complication was that only cells co-transfected with Cas9 and gRNA plasmids were mutants
To circumvent this limitation and facilitate delivery of all components through a single construct, we explored alternative strategies to transcribe gRNAs that eliminated the requirement for a U6 promoter, thereby enabling Cas9 and gRNA production via the same promoter

Summary

Introduction

Advent of CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) is a highlight of the past decade that has enabled genome editing across multiple species (Chen et al, 2013; Cong et al, 2013; Dickinson et al, 2013; Gagnon et al, 2014; Stolfi et al, 2014). Conventional molecular perturbation studies in chick embryos have relied upon electroporation of dominant-negative transgenes, antisense morpholinos or short-hairpin RNAs (Corey and Abrams, 2001; Sauka-Spengler and Barembaum, 2008) and, more recently, Cas and guide RNAs (gRNAs) (Gandhi et al, 2017; Williams et al, 2018). A major drawback of the latter approach was that Cas and gRNAs were delivered through separate constructs. Only those cells co-transfected with both plasmids were mutants, these could not be distinguished from cells transfected with the fluorescent marker alone. The absence of permanent labeling compromised the ability to identify or trace the descendants of individually perturbed cells

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Conclusion