Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
Proc Natl Acad Sci U S A. 2013 Aug 27;110(35):14396-401. doi: 10.1073/pnas.1300108110. Epub 2013 Aug 12.
Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins constitute a recently identified prokaryotic defense system against invading nucleic acids. DNA segments, termed protospacers, are integrated into the CRISPR array in a process called adaptation. Here, we establish a PCR-based assay that enables evaluating the adaptation efficiency of specific spacers into the type I-E Escherichia coli CRISPR array. Using this assay, we provide direct evidence that the protospacer adjacent motif along with the first base of the protospacer (5'-AAG) partially affect the efficiency of spacer acquisition. Remarkably, we identified a unique dinucleotide, 5'-AA, positioned at the 3' end of the spacer, that enhances efficiency of the spacer's acquisition. Insertion of this dinucleotide increased acquisition efficiency of two different spacers. DNA sequencing of newly adapted CRISPR arrays revealed that the position of the newly identified motif with respect to the 5'-AAG is important for affecting acquisition efficiency. Analysis of approximately 1 million spacers showed that this motif is overrepresented in frequently acquired spacers compared with those acquired rarely. Our results represent an example of a short nonprotospacer adjacent motif sequence that affects acquisition efficiency and suggest that other as yet unknown motifs affect acquisition efficiency in other CRISPR systems as well.
成簇规律间隔短回文重复序列（CRISPR）及其相关蛋白构成了一种最近发现的原核防御系统，用于抵御入侵的核酸。被称为前导序列的 DNA 片段通过一个称为适应的过程整合到 CRISPR 阵列中。在这里，我们建立了一种基于 PCR 的检测方法，用于评估特定间隔子在 I-E 型大肠杆菌 CRISPR 阵列中的适应效率。使用该检测方法，我们提供了直接证据表明，前导序列相邻基序和前导序列的第一个碱基（5'-AAG）部分影响间隔子获取的效率。值得注意的是，我们鉴定出一种独特的二核苷酸，即 5'-AA，位于间隔子的 3' 端，增强了间隔子获取的效率。插入该二核苷酸可增加两个不同间隔子的获取效率。对新适应的 CRISPR 阵列的 DNA 测序表明，新鉴定的基序相对于 5'-AAG 的位置对于影响获取效率很重要。对大约 100 万个间隔子的分析表明，与很少被获取的间隔子相比，这个新的基序在经常被获取的间隔子中出现的频率更高。我们的研究结果代表了一个影响获取效率的短非前导序列相邻基序序列的例子，并表明其他尚未被发现的基序也会影响其他 CRISPR 系统的获取效率。
