Efficient and rapid fluorescent protein knock-in with universal donors in mammalian stem cells

Shi, Y.; Kopparapu, N.; Ohler, L.; Dickinson, D. J.

doi:10.1101/2022.10.10.511307

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bioRxiv · 10.1101/2022.10.10.511307

Efficient and rapid fluorescent protein knock-in with universal donors in mammalian stem cells

Shi, Y.·Kopparapu, N.·Ohler, L.·Dickinson, D. J.

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Abstract

Fluorescent protein (FP) tagging is a foundational approach in cell biology because it allows observation of protein distribution, dynamics, and interaction with other proteins in living cells. However, the typical approach using overexpression of tagged proteins can perturb cell behavior and introduce localization artifacts. To preserve native expression, fluorescent proteins can be inserted directly into endogenous genes. This approach has been standard in yeast for decades, and more recently in invertebrate model organisms with the advent of CRISPR/Cas9. However, endogenous fluorescent protein tagging has not been widely used in mammalian cells due to inefficient homology-directed repair (HDR). Here, we describe a streamlined method for efficient and fast integration of FP tags into native loci via non-homologous end joining (NHEJ) in mouse embryonic stem cells. Our protocol minimizes cloning with universal donors, allows for N or C-terminal tagging of endogenous proteins, and requires less than 2 weeks from transfection to imaging, thereby improving the applicability of FP knock-in in mammalian cells.

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Shi, Y., Kopparapu, N., Ohler, L., Dickinson, D. J.. 2022-10-11. Efficient and rapid fluorescent protein knock-in with universal donors in mammalian stem cells. https://doi.org/10.1101/2022.10.10.511307

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