PDF(Pubmed)
Abstract：
The CRISPR/Cas9 technique applied to modify the cattle genome has value in increasing animal health and welfare. Here, we established a simple, fast, and efficient cloning-free CRISPR/Cas9 protocol for large deletions of genomic loci in the frequently used model bovine MDBK cell line. The main advantages of our protocol are as follows: (i) pre-screening of the sgRNA efficiency with a fast and simple cleavage assay, (ii) reliable detection of genomic edits primarily by PCR and confirmed by DNA sequencing, and (iii) single cell sorting with FACS providing specific genetic information from modified cells of interest. Therefore, our method could be successfully applied in different studies, including functional validation of any genetic or regulatory elements.
摘要：
用于修改牛基因组的CRISPR/Cas9技术在增加动物健康和福利方面具有价值。这里,我们建立了一个简单的，快,和有效的无克隆CRISPR/Cas9方案，用于在常用的模型牛MDBK细胞系中大量缺失基因组基因座。我们的方案的主要优点如下：（i）用快速简单的切割测定预先筛选sgRNA效率，（ii）可靠的检测基因组编辑主要通过PCR和DNA测序证实，和(iii)用FACS进行单细胞分选,提供来自感兴趣的修饰细胞的特定遗传信息。因此,我们的方法可以成功地应用于不同的研究，包括任何遗传或调控元件的功能验证。
