Carlota Ramos

Precise analysis of endogenous protein behavior in mosaic tissues requires strategies that both modify native alleles and identify successfully edited cells at single-cell resolution. Here, we establish a CRISPR-based genome-editing platform that combines endogenous protein visualization with allele-specific mutant tracking in vivo. Using the chick neural tube and β-catenin as a model system, we validate cytosine base editing, prime editing, and twin-prime editing by introducing stabilizing mutations that reproduce their expected cellular and morphological phenotypes. We then develop a twin-prime editing strategy that couples installation of a defined oncogenic mutation to simultaneous insertion of minimal peptide tags, thereby making productive editing directly observable at single-cell resolution without clonal selection. Comparative analysis of ALFA, V5, and split-GFP tags identifies split-GFP as the most reliable strategy for endogenous protein visualization in vivo, and fluorescence-based cell selection strongly enriches for the intended twin-prime editing product. Finally, we extend the approach to endogenous wild-type β-catenin, identifying a functionally neutral insertion site that enables visualization at physiological, non-stabilized levels while preserving normal protein behavior. Together, these results establish twin-prime editing as a versatile platform for directly linking precise endogenous genome modification to protein visualization and allele-specific mutation analysis in intact vertebrate tissues.

Vinculin is an actin-binding protein (ABP) that strengthens the connection between the actin cytoskeleton and adhesion complexes. It binds to β-catenin/N-cadherin complexes in apical adherens junctions (AJs), which maintain cell-to-cell adhesions, and to talin/integrins in the focal adhesions (FAs) that attach cells to the basal membrane. Here, we demonstrate that β-catenin targets vinculin to the apical AJs and the centrosome in the embryonic neural tube (NT). Suppression of vinculin slows down the basal-to-apical part of interkinetic nuclear migration (BAINM), arrests neural stem cells (NSCs) in the G2 phase of the cell cycle, and ultimately dismantles the apical actin cytoskeleton. In the NSCs, mitosis initiates when an internalized centrosome gathers with the nucleus during BAINM. Notably, our results show that the first centrosome to be internalized is the daughter centrosome, where β-catenin and vinculin accumulate, and that vinculin suppression prevents centrosome internalization. Thus, we propose that vinculin links AJs, the centrosome, and the actin cytoskeleton where actomyosin contraction forces are required.