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

## TL;DR

This study improves the efficiency of gene editing in sheep cells by using a CRISPR/Cas9-Gal4 system to enhance targeted DNA insertion.

## Contribution

A novel CRISPR/Cas9-Gal4 system is introduced to increase gene knock-in efficiency in sheep cells without drug selection.

## Key findings

- Knock-in efficiency improved from 5.30% to 16.67% in sheep fetal fibroblasts.
- The Gal4 system enhances local concentration of the repair template at DNA breaks.
- The method is efficient and user-friendly for CRISPR-mediated gene editing in sheep.

## Abstract

Currently, a major challenge exists in CRISPR-mediated genome editing research in sheep: the low efficiency of exogenous large DNA fragment targeted integration without drug selection or fluorescence enrichment. This restriction significantly impedes the use of precise genome editing in sheep for agricultural, biological, and biomedical purposes. In this study, we employed the strategy of increasing the local concentration of the homologous repair template at the site of the DNA double-strand break (DSB). We achieved this by fusing the DNA binding domain (BD) of the Gal4 protein (Gal4-BD) to the N-terminal end of the SpCas9 protein using a 32-amino acid (aa) flexible linker. Additionally, we incorporated a 17 bp UAS at the 3′ end of the donor template, which can be specifically recognized and bound by Gal-BD. As a result, we observed a significant improvement in the knock-in efficiency of the exogenous large DNA fragment (2997 bp) in sheep fetal fibroblasts (SFFs), increasing it from 5.30% (8/151) to 16.67% (32/192), providing a comparatively efficient and user-friendly method to promote CRISPR-mediated gene knock-in in sheep.