背景:基于基因组编辑系统,基因敲除和敲入已在大型农场动物中广泛进行。然而,许多类型的精确基因编辑,包括有针对性的删除,基因标记,和大的基因片段替换,在大型农场动物中仍然是一个挑战。
结果:这里,我们建立了多功能的自切除基因打靶技术与可编程核酸酶(SEGCPN)相结合,以有效地在牛中产生各种类型的精确基因编辑。首先,我们使用这种通用方法成功地生成了在MSTN基因座上具有点突变和11bp缺失的牛胚胎。第二,我们成功地产生了在SRY基因座上有EGFP标记的公牛。最后,我们成功产生了人源化奶牛,其中内源性18kbα-酪蛋白基因被2.6kb人α-乳清蛋白基因取代。
结论:总之,我们新的SEGCPN方法为大型动物中各种类型的精确基因编辑提供了无限的可能性,适用于农业和疾病模型。
Gene knockout and knock-in have been widely performed in large farm animals based on genome editing systems. However, many types of precise gene editing, including targeted deletion, gene tagging, and large gene fragment replacement, remain a challenge in large farm animals.
Here, we established versatile self-excising gene-targeting technology in combination with programmable nucleases (SEGCPN) to efficiently generate various types of precise gene editing in bovine. First, we used this versatile method to successfully generate bovine embryos with point mutations and 11-bp deletions at the MSTN locus. Second, we successfully generated bulls with EGFP labeling at the SRY locus. Finally, we successfully generated humanized cows in which the endogenous 18-kb α-casein gene was replaced with a 2.6-kb human α-lactalbumin gene.
In summary, our new SEGCPN method offers unlimited possibilities for various types of precise gene editing in large animals for application both in agriculture and disease models.