CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism.

Area of Science:

• Genetics
• Molecular Biology
• Genome Engineering

Background:

• Studying gene function requires precise genome modification.
• Existing methods for inserting large DNA fragments can be inefficient.
• CRISPR-Cas9 technology offers potential for targeted genome editing.

Purpose of the Study:

• To develop a novel system for efficient, site-specific insertion of large DNA cassettes into eukaryotic genomes.
• To enable flexible C-terminal tagging of endogenous proteins.
• To characterize the DNA repair pathways involved in this insertion process.

Main Methods:

• Utilized the CRISPR-Cas9 system to create targeted double-strand breaks (DSBs).
• Developed a modular system (CRISPaint) for integrating universal donor DNA at DSB sites.
• Employed minicircle DNA as an optional donor format.
• Investigated the role of DNA repair components (DNA-PKcs, XLF, ligase-4) in site-specific insertion.

Main Results:

• CRISPaint achieved precise and efficient integration of large DNA cassettes into eukaryotic genomes.
• The system successfully created C-terminal tag fusions of endogenous proteins in human cells with high efficiency.
• Site-specific insertion was found to be entirely dependent on canonical Non-Homologous End Joining (NHEJ) repair.

Conclusions:

• CRISPaint is a versatile and efficient tool for site-specific genome engineering.
• The system facilitates the study of gene function through precise protein tagging.
• Understanding the reliance on NHEJ provides insights into genome repair mechanisms.