Vandemoortele Giel, De Sutter Delphine, Eyckerman Sven
VIB-UGent Center for Medical Biotechnology, Ghent, Belgium.
Department of Biochemistry, Ghent University, Ghent, Belgium.
Bio Protoc. 2017 Apr 5;7(7):e2211. doi: 10.21769/BioProtoc.2211.
The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair mechanism. However, in presence of a DNA repair template, Homology-Directed Repair (HDR) can occur leading to precise repair of the lesion site. This last process can be exploited to enable precise knock-in changes by introducing the desired genomic alteration on the repair template. In this protocol, we describe the delivery of long repair templates (> 200 nucleotides) using recombinant Adeno Associated Virus (rAAV) for CRISPR-Cas9-based knock-in of a C-terminal tag sequence in a human cell line.
可编程的成簇规律间隔短回文重复序列（CRISPR）相关核酸酶9（Cas9）技术通过提供一种在所需位置切割基因组的有效方法，彻底改变了基因组编辑（莱德福德，2015年）。在哺乳动物细胞中，DNA损伤会触发易出错的非同源末端连接（NHEJ）DNA修复机制。然而，在存在DNA修复模板的情况下，同源定向修复（HDR）可能会发生，从而导致损伤位点的精确修复。通过在修复模板上引入所需的基因组改变，可以利用这一过程实现精确的敲入改变。在本方案中，我们描述了使用重组腺相关病毒（rAAV）递送长修复模板（>200个核苷酸），用于在人细胞系中基于CRISPR-Cas9的C末端标签序列敲入。
