Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system

Area of Science:

• Genetics
• Molecular Biology
• Gene Editing Technologies

Background:

• CRISPR-Cas9 is widely used for gene targeting.
• Existing methods often produce small indels, leading to multiple mutations and alleles.
• Efficient generation of large genomic deletions is challenging.

Purpose of the Study:

• To develop and validate a dual sgRNA-directed CRISPR/Cas9 system for generating large-fragment deletions.
• To investigate the efficiency and heritability of large deletions in rabbits.
• To assess the impact of large deletions on gene expression and phenotype.

Main Methods:

• Utilized a dual single-guide RNA (sgRNA) strategy with the CRISPR/Cas9 system.
• Targeted the tyrosinase (TYR) gene in rabbits to induce a 105 kb deletion.
• Analyzed gene expression, phenotype (albinism), and germline transmission in offspring.

Main Results:

• Successfully generated a 105 kb deletion in the rabbit TYR gene.
• Observed typical albinism symptoms and significantly decreased TYR gene expression in knockout rabbits.
• Demonstrated stable inheritance of the large deletion and albinism phenotype in the F1 generation.
• Confirmed the absence of mosaic mutations and off-target effects.

Conclusions:

• The dual sgRNA-directed CRISPR/Cas9 system enables efficient mono- and biallelic large-fragment deletions.
• This method is effective for generating targeted large deletions with stable germline transmission.
• The system offers a precise and reliable tool for genetic engineering in rabbits and potentially other species.