CRISPR-mediated Labeling of Cells in Chick Embryos Based on Selectively Expressed Genes

Area of Science:

• Developmental Biology
• Genetics
• Neuroscience

Background:

• Precise cell labeling and manipulation are crucial for functional, structural, molecular, and developmental analyses in model organisms.
• While germline transgenesis works in some species, alternative methods are needed for selective somatic cell labeling based on gene expression in others.

Purpose of the Study:

• To describe a novel CRISPR-based method for somatic integration of reporters or Cre recombinase into specific genes in the chick genome.
• To enable visualization of cells in the retina and midbrain using this new technique.

Main Methods:

• CRISPR-based somatic integration of reporters or Cre recombinase into targeted genes in the chick genome.
• Utilizing an RNA-seq-based cell atlas to select specific genomic loci for editing.
• Electroporation and CRISPR-mediated homology-instructed knock-in (eCHIKIN) for precise gene modification.
• Reporters designed for soluble visualization of cell morphology or appended to proteins for subcellular localization studies.

Main Results:

• Successful implementation of the eCHIKIN method for targeted gene editing in chick somatic cells.
• Visualization of specific cell populations within the retina and midbrain.
• Demonstration of both soluble reporters for cell morphology and protein-appended reporters for subcellular localization.

Conclusions:

• The eCHIKIN method provides a powerful new tool for functional, structural, molecular, and developmental analyses in chick model organisms.
• This technique allows for precise genetic manipulation and visualization of specific cell types in vivo.
• eCHIKIN expands the toolkit for studying gene function and cell biology in non-traditional model systems.