用CRISPR-Cas9在绵羊成纤维细胞ACTG1导入荧光标记基因

【目的】在绵羊成纤维细胞中,针对ACTG1基因羧基端,定点导入荧光蛋白标记基因,将外源基因定点导入绵羊基因组中,建立有效的方法。【方法】CRISPR-Cas9系统在绵羊成纤维细胞基因组特定区域引起DNA双链断裂,从而诱导细胞修复断裂的的基因组。通过NHEJ修复途径,在特定位点导入外源基因,改善定点导入效率。【结果】采用CRISPaint通用供体模板,结合CRISPR-Cas9系统,在绵羊成纤维细胞ACTG1基因导入荧光标记效率达1.4%,获得了外源基因定点导入的单克隆细胞株。【结论】CRISPR-Cas9系统结合NHEJ,能够有效的将大的外源DNA序列导入绵羊成纤维细胞的预定基因组位点。

Use of the CRISPR-Cas9 System in Sheep Fibroblasts to Lead Fluorescent Tags into ACTG1 Gene

【Objective】 This project aims to establish a method for leading foreign genes into the specific site of sheep fibroblast genome, hoping applying this method allows creating C-terminal tag fusions of endogenously encoded proteins in sheep ACTG1 gene with high efficiencies.【Method】 The CRISPR-Cas9 system caused double-strand to break in specific regions of cells, thus inducing cells to repair broken genomes, and the non-homologous end joining (NHEJ) repair pathway was used to improve knock-in efficiency of the exogenous gene.【Result】 The fluorescent protein open reading frame (ORF) was inserted into ACTG1 gene of sheep fibroblasts by the CRISPaint universal donor and CRISPR-Cas9 system. The insertion efficiency of exogenous gene achieved 1.4% of the total cells. Monoclonal cell lines were obtained for site-specific introduction of foreign genes.【Conclusion】 This technique holds promise for quick and efficient insertion of a large foreign DNA sequence into a predetermined genomic site in sheep fibroblasts by CRISPR-Cas9 system and NHEJ repair pathway.