Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):E2579-86. doi: 10.1073/pnas.1208507109. Epub 2012 Sep 4.
Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a double-strand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites, RuvC and HNH, to generate site-specific nicks on opposite DNA strands. Results demonstrate that the Cas9-crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and protein-mediated DNA cleavage. These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.
成簇规律间隔短回文重复（CRISPR）/CRISPR 相关（Cas）系统为细菌和古菌提供了针对病毒和质粒的适应性免疫。通过预先加载有小的、干扰性 CRISPR RNA（crRNA）的核糖核蛋白复合物来执行对入侵核酸的沉默，这些 crRNA 充当靶向和降解外源核酸的向导。在这里，我们证明了嗜热链球菌 CRISPR3/Cas 系统的 Cas9-crRNA 复合物在含有与 crRNA 互补序列的 DNA 特定位点引入双链断裂。DNA 切割由 Cas9 执行，Cas9 使用两个不同的活性位点 RuvC 和 HNH，在相反的 DNA 链上产生特异性的切口。结果表明，Cas9-crRNA 复合物作为一种 RNA 指导的内切酶，具有 RNA 指导的靶序列识别和蛋白介导的 DNA 切割功能。这些发现为工程化通用可编程 RNA 引导的 DNA 内切酶铺平了道路。
