Endogenous Fluorescence Tagging by CRISPR

Area of Science:

• Biomedical Research
• Molecular Biology
• Genetics

Background:

• Fluorescent proteins enable live cell imaging without fixation.
• Current methods facilitate rapid integration of fluorescent markers into endogenous genes.

Purpose of the Study:

• To review gene editing technologies for fluorescent protein knock-ins.
• To focus on CRISPR-Cas9 strategies for marker integration.
• To discuss advancements and future perspectives in fluorescent protein applications.

Main Methods:

• Overview of zinc finger nucleases (ZFNs), transcription activator-like effectors (TALENs), and CRISPR.
• Detailed examination of CRISPR-based strategies: homology-directed repair (HDR), microhomology-mediated end joining (MMEJ), and nonhomologous end joining (NHEJ).
• Analysis of recent developments for highly efficient knock-in procedures.

Main Results:

• CRISPR technology offers versatile strategies (HDR, MMEJ, NHEJ) for fluorescent marker knock-in.
• Recent advancements have significantly improved the efficiency of gene editing for marker integration.
• Fluorescent protein knock-ins offer unique advantages for live cell imaging and biological studies.

Conclusions:

• CRISPR technology is a powerful tool for fluorescent protein knock-ins, advancing live cell imaging.
• Future directions include combining gene editing with organoid development for enhanced research.
• Efficient marker integration via CRISPR opens new avenues in biomedical research.