Trevino Alexandro E, Zhang Feng
Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Methods Enzymol. 2014;546:161-74. doi: 10.1016/B978-0-12-801185-0.00008-8.
The RNA-guided, sequence-specific endonuclease Cas9 has been widely adopted as genome engineering tool due to its efficiency and ease of use. Derived from the microbial CRISPR (clustered regularly interspaced short palindromic repeat) type II adaptive immune system, Cas9 has now been successfully engineered for genome editing applications in a variety of animal and plant species. To reduce potential off-target mutagenesis by wild-type Cas9, homology- and structure-guided mutagenesis of Streptococcus pyogenes Cas9 catalytic domains has produced "nicking" enzymes (Cas9n) capable of inducing single-strand nicks rather than double-strand breaks. Since nicks are generally repaired with high fidelity in eukaryotic cells, Cas9n can be leveraged to mediate highly specific genome editing, either via nonhomologous end-joining or homology-directed repair. Here we describe the preparation, testing, and application of Cas9n reagents for precision mammalian genome engineering.
RNA引导的序列特异性核酸内切酶Cas9因其高效性和易用性，已被广泛用作基因组工程工具。Cas9源自微生物CRISPR（成簇规律间隔短回文重复序列）II型适应性免疫系统，目前已成功改造，可用于多种动植物物种的基因组编辑应用。为减少野生型Cas9潜在的脱靶诱变，化脓性链球菌Cas9催化结构域的同源性和结构引导诱变产生了能够诱导单链切口而非双链断裂的“切口”酶（Cas9n）。由于在真核细胞中，切口通常以高保真度修复，因此Cas9n可用于通过非同源末端连接或同源定向修复来介导高度特异性的基因组编辑。在此，我们描述了用于精确哺乳动物基因组工程的Cas9n试剂的制备、测试和应用。
