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

Fluorescent protein (FP) tagging isa key method for observing 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 widely used in yeast for decades, and more recently in invertebrate model organisms with the advent of CRISPR/Cas9. However, endogenous FP tagging has not been widely used in mammalian cells due to inefficient homology-directed repair. Recently, the CRISPaint system used non-homologous end joining for efficient integration of FP tags into native loci, but it only allows C-terminal knock-ins. Here, we have enhanced the CRISPaint system by introducing new universal donors for N-terminal insertion and for multi-color tagging with orthogonal selection markers. We adapted the procedure for mouse embryonic stem cells, which can be differentiated into diverse cell types. Our protocol is rapid and efficient, enabling live imaging in less than 2 weeks post-transfection. These improvements increase the versatility and applicability of FP knock-in in mammalian cells.

Automated summary

Fluorescent protein tagging is a crucial method for studying protein behavior in living cells. However, overexpression of tagged proteins can disrupt cell behavior. This study presents an improved method for inserting fluorescent proteins directly into native genes in mammalian cells using the CRISPaint system. The protocol is efficient and enables live imaging in less than two weeks post-transfection, increasing the versatility of fluorescent protein knock-in.