CRISPR-Cas9 系统中 RNA 指导的核糖核酸酶活性的生化特性分析。
Biochemical characterization of RNA-guided ribonuclease activities for CRISPR-Cas9 systems.
机构信息
Department of Biological Chemistry, University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA.
Department of Biological Chemistry, University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA.
出版信息
Methods. 2020 Feb 1;172:32-41. doi: 10.1016/j.ymeth.2019.06.018. Epub 2019 Jun 20.
The majority of bacteria and archaea rely on CRISPR-Cas systems for RNA-guided, adaptive immunity against mobile genetic elements. The Cas9 family of type II CRISPR-associated DNA endonucleases generates programmable double strand breaks in the CRISPR-complementary DNA targets flanked by the PAM motif. Nowadays, CRISPR-Cas9 provides a set of powerful tools for precise genome manipulation in eukaryotes and prokaryotes. Recently, a few Cas9 orthologs have been reported to possess intrinsic CRISPR-guided, sequence-specific ribonuclease activities. These discoveries fundamentally expanded the targeting capability of CRISPR-Cas9 systems, and promise to provide new CRISPR tools to manipulate specific cellular RNA transcripts. Here we present a detailed method for the biochemical characterization of Cas9's RNA-targeting potential.
大多数细菌和古菌依赖 CRISPR-Cas 系统来实现针对移动遗传元件的 RNA 导向、适应性免疫。Cas9 家族的 II 型 CRISPR 相关 DNA 内切酶在 PAM 基序侧翼的 CRISPR 互补 DNA 靶标上产生可编程双链断裂。如今，CRISPR-Cas9 为真核生物和原核生物的精确基因组操作提供了一组强大的工具。最近，有报道称一些 Cas9 同源物具有内在的 CRISPR 引导、序列特异性核糖核酸酶活性。这些发现从根本上扩展了 CRISPR-Cas9 系统的靶向能力，并有望提供新的 CRISPR 工具来操纵特定的细胞 RNA 转录物。在这里，我们介绍了一种用于 Cas9 的 RNA 靶向潜力的生化特性分析的详细方法。
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