Labeling Endogenous Proteins Using CRISPR-mediated Insertion of Exon (CRISPIE)

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 technology revolutionized eukaryotic genome editing.
• Precise knock-in of large DNA sequences, like fluorescent proteins, remains challenging, especially in post-mitotic cells.
• Existing methods often yield insertions/deletions (INDELs) due to non-homologous end joining (NHEJ).

Purpose of the Study:

• To develop a method for precise large DNA sequence insertion into eukaryotic genomes, overcoming INDEL errors.
• To enable efficient gene labeling and modification in challenging cell types.
• To provide a versatile tool for various gene editing applications.

Main Methods:

• Developed CRISPR-mediated insertion of exon (CRISPIE) technology.
• CRISPIE inserts a designer exon into an intronic location, flanked by intron sequences.
• Utilized the splicing mechanism to remove potential INDELs at the insertion junction.

Main Results:

• CRISPIE achieves virtually error-free mRNA transcripts by splicing out INDELs.
• Demonstrated high-efficiency endogenous labeling of β-actin with EGFP in human cells.
• Showcased compatibility with N- and C-terminal labels and various transfection methods.
• Incorporated optional removal of protein modification via exogenous sgRNA sites.

Conclusions:

• CRISPIE overcomes limitations of traditional CRISPR knock-ins, enabling precise large DNA insertions.
• The method facilitates efficient in vivo gene labeling, applicable to post-mitotic neurons.
• CRISPIE offers a robust and adaptable platform for diverse gene editing applications.