Kaulich Manuel, Dowdy Steven F
Department of Cellular and Molecular Medicine, University of California , San Diego, La Jolla, California.
Nucleic Acid Ther. 2015 Dec;25(6):287-96. doi: 10.1089/nat.2015.0545. Epub 2015 Nov 5.
Altering endogenous genes in cells is an integral tool of modern cell biology. The ease-of-use of the CRISPR/Cas9 system to introduce genomic DNA breaks at specific sites in vivo has led to its rapid and wide adoption. In the absence of a DNA template, the lesion is repaired by nonhomologous end joining resolving as internal deletions. However, in the presence of a homologous DNA template, homology-directed repair occurs with variable efficiencies. Recent work has demonstrated that highly efficient gene targeting can be induced by combining CRISPR/Cas9 targeting of genomic loci with recombinant adeno-associated virus (rAAV) to provide a single-stranded homologous DNA template. Here we review the current state of CRISPR/Cas-based gene editing and provide a practical guide to applying the CRISPR/Cas and rAAV system for highly efficient, time- and cost-effective gene targeting.
改变细胞中的内源基因是现代细胞生物学不可或缺的工具。CRISPR/Cas9系统能够在体内特定位点引入基因组DNA断裂，因其使用便捷，已迅速得到广泛应用。在没有DNA模板的情况下，损伤通过非同源末端连接修复，结果是内部缺失。然而，在存在同源DNA模板的情况下，同源定向修复的效率各不相同。最近的研究表明，通过将基因组位点的CRISPR/Cas9靶向与重组腺相关病毒（rAAV）相结合，以提供单链同源DNA模板，可以诱导高效的基因靶向。在此，我们综述了基于CRISPR/Cas的基因编辑的现状，并提供了一份实用指南，介绍如何应用CRISPR/Cas和rAAV系统进行高效、省时且经济高效的基因靶向操作。
