Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel.
Nucleic Acids Res. 2012 Jul;40(12):5569-76. doi: 10.1093/nar/gks216. Epub 2012 Mar 8.
The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called 'adaptation', (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.
成簇规律间隔短回文重复序列及其相关蛋白（CRISPR/Cas）构成了一种最近发现的原核防御机制，可抵抗入侵的核酸。CRISPR/Cas 系统的活性包括三个步骤：（i）将外源 DNA 序列插入 CRISPR 阵列中，以防止未来的攻击，这一过程称为“适应”；（ii）相关蛋白的表达，以及阵列的表达和加工，随后是（iii）RNA 介导的对外源核酸的干扰。在这里，我们描述了一种在大肠杆菌中强大的检测方法，以探索迄今为止研究最少的适应过程。我们确定了在先导序列和阵列中对适应步骤至关重要的必需基因和 DNA 元件。我们还通过显示第一个重复序列作为新插入重复序列的模板，提供了关于重复-间隔单元插入的机制见解。总之，我们的结果阐明了 CRISPR/Cas 系统适应过程的基本步骤。
