CRISPR-induced exon skipping is dependent on premature termination codon mutations

Area of Science:

• Molecular Biology
• Genetics
• Gene Editing Technologies

Background:

• CRISPR/Cas9 gene editing is known to cause unintended exon skipping.
• The precise mechanism driving CRISPR-mediated exon skipping remains debated.
• Previous research indicates a link between gene editing and splicing alterations.

Purpose of the Study:

• To investigate the mechanism of CRISPR/Cas9-induced exon skipping.
• To determine the specific mutation types associated with high-frequency exon skipping.
• To clarify the role of nonsense-associated altered splicing in this phenomenon.

Main Methods:

• Analysis of 22 gene-edited rabbit lines created using CRISPR/Cas9.
• Comparative assessment of exon skipping frequency across different mutation types (premature termination codon, non-frameshift, missense).
• Evaluation of splicing patterns in relation to mutation location and type.

Main Results:

• High-frequency exon skipping was observed specifically in rabbits with premature termination codon (PTC) mutations.
• No significant exon skipping was detected in rabbits with mutations in exon 1, non-frameshift mutations, or missense mutations.
• The findings indicate a strong correlation between PTC mutations and CRISPR-mediated exon skipping.

Conclusions:

• CRISPR-mediated exon skipping is significantly influenced by the presence of premature termination codon mutations.
• The study provides evidence that nonsense-associated altered splicing is the underlying mechanism for CRISPR-induced exon skipping in PTC-mutated rabbits.
• This research clarifies a controversial aspect of CRISPR/Cas9 technology, highlighting mutation-specific effects on splicing.