A simple and efficient method to visualize and quantify the efficiency of chromosomal mutations from genome editing

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Genome editing technologies like TALEN and CRISPR/Cas enable precise genetic modifications.
• Accurate characterization of induced mutations is crucial for assessing genome-editing efficacy and specificity.
• Existing mutation identification methods, primarily sequencing, are not always optimal for identifying gene-disrupting indels.

Purpose of the Study:

• To develop a simple and efficient method for visualizing and quantifying genome editing-induced mutations in vivo.
• To facilitate the screening of effective genome-editing enzymes and identification of desired mutant cells/animals.

Main Methods:

• A novel reporter system utilizing a two-color fusion protein expressed from a vector.
• The vector allows insertion of the edited genomic region between two color moieties (e.g., LacZα and GFP).
• Analysis of mutation rates through bright field microscopy, eliminating the need for fluorescence.

Main Results:

• The method successfully visualizes and quantifies genome editing efficiency in vivo.
• It enables straightforward identification of animals with desired mutations (out-of-frame indels).
• The system, using LacZα and GFP, allows analysis without a fluorescent microscope, simplifying the process.

Conclusions:

• This novel reporter system provides a simple, efficient, and cost-effective way to assess genome editing outcomes.
• It significantly streamlines the process of screening genome editing tools and identifying targeted mutations.
• The approach enhances the ability to rapidly identify and select desired mutant cells or organisms.