PDF(Pubmed)
Abstract：
The CRISPR/Cas9 system is widely used for genome editing in livestock production, although off-target effects can occur. It is the main method to produce genome-edited goats by somatic cell nuclear transfer (SCNT) of CRISPR/Cas9-mediated genome-edited primary goat fetal fibroblast cells (GFFs). Improving the double-strand break (DSB) efficiency of Cas9 in primary cells would improve the homologous repair (HR) efficiency. The low efficiency of HR remains a major hurdle in CRISPR/Cas9-mediated precise genome editing, increasing the work required to screen the genome-edited primary cell clones. In this study, we modified several essential parameters that affect the efficiency of the CRISPR/Cas9-mediated knock-in GFF cloning system, including establishing a high-efficiency transfection system for primary cells via nucleofection and optimizing homology arm (HA) length during HR. Here, we specifically inserted a recombinant human butyrylcholinesterase gene (rhBChE) into the goat fibroblast growth factor (FGF)-5 locus through the CRISPR/Cas9 system, thereby achieving simultaneous rhBChE insertion and FGF5 knock-out. First, this study introduced the Cas9, FGF5 knock-out small guide RNA, and rhBChE knock-in donors into GFFs by electroporation and obtained positive cell clones without off-target effects. Then, we demonstrated the expression of rhBChE in GFF clones and verified its function. Finally, we obtained a CRISPR/Cas9-mediated rhBChE-overexpression goat.
摘要：
CRISPR/Cas9系统广泛用于畜牧业生产中的基因组编辑，尽管可能会发生脱靶效应。它是通过CRISPR/Cas9介导的基因组编辑的原代山羊胎儿成纤维细胞（GFFs）的体细胞核移植（SCNT）生产基因组编辑的山羊的主要方法。提高Cas9在原代细胞中的双链断裂(DSB)效率将提高同源修复(HR)效率。HR的低效率仍然是CRISPR/Cas9介导的精确基因组编辑的主要障碍。增加筛选基因组编辑的原代细胞克隆所需的工作。在这项研究中,我们修改了影响CRISPR/Cas9介导的敲入GFF克隆系统效率的几个基本参数，包括通过核转染为原代细胞建立高效转染系统，并在HR期间优化同源臂（HA）长度。这里,我们通过CRISPR/Cas9系统将重组人丁酰胆碱酯酶基因（rhBChE）插入山羊成纤维细胞生长因子（FGF）-5位点，从而同时实现rhBChE插入和FGF5敲除。首先,本研究引入了Cas9，FGF5敲除小指导RNA，和rhBChE通过电穿孔将供体敲入GEF并获得阳性细胞克隆而没有脱靶效应。然后，我们证明了rhBChE在GFF克隆中的表达并验证了其功能。最后,我们获得了CRISPR/Cas9介导的rhBChE过表达山羊。
