Improving Gene Knock-In Efficiencies in Sheep Primary Cells Using a CRISPR/Cas9-Gal4 System.

Area of Science:

• * Molecular Biology
• * Genetics
• * Biotechnology

Background:

• * CRISPR-mediated genome editing in sheep faces challenges with low efficiency for large DNA fragment integration.
• * This limitation hinders precise gene editing applications in sheep for agriculture, biology, and biomedicine.
• * Current methods often rely on drug selection or fluorescence enrichment, which are not always ideal.

Purpose of the Study:

• * To enhance the efficiency of targeted integration of large exogenous DNA fragments in sheep using CRISPR technology.
• * To develop a more user-friendly and efficient method for CRISPR-mediated gene knock-in in sheep.
• * To overcome the limitations of low knock-in efficiency in sheep fetal fibroblasts.

Main Methods:

• * Employed a strategy to increase the local concentration of the homologous repair template at the DNA double-strand break (DSB) site.
• * Fused the DNA binding domain (BD) of the Gal4 protein (Gal4-BD) to the N-terminal end of the SpCas9 protein via a flexible linker.
• * Incorporated a UAS sequence at the 3' end of the donor template for specific binding by Gal4-BD.

Main Results:

• * Achieved a significant improvement in the knock-in efficiency of a large exogenous DNA fragment (2997 bp).
• * Knock-in efficiency in sheep fetal fibroblasts (SFFs) increased from 5.30% (8/151) to 16.67% (32/192).
• * Demonstrated a user-friendly method for efficient CRISPR-mediated gene knock-in in sheep.

Conclusions:

• * The developed method effectively enhances the efficiency of large DNA fragment knock-in in sheep.
• * This strategy offers a valuable tool for advancing CRISPR-mediated genome editing in sheep.
• * The findings support broader applications of precise gene editing in sheep for various scientific and agricultural purposes.