The adaptive CRISPR-Cas immune system stores sequences from past invaders as spacers in CRISPR arrays and thereby provides direct evidence that links invaders to hosts. Mapping CRISPR spacers has revealed many aspects of CRISPR-Cas biology, including target requirements such as the protospacer adjacent motif (PAM). However, studies have so far been limited by a low number of mapped spacers in the database. By using vast metagenomic sequence databases, we map approximately one-third of more than 200,000 unique CRISPR spacers from a variety of microbes and derive a catalog of more than two hundred unique PAM sequences associated with specific CRISPR-Cas subtypes. These PAMs are further used to correctly assign the orientation of CRISPR arrays, revealing conserved patterns between the last nucleotides of the CRISPR repeat and PAM. We could also deduce CRISPR-Cas subtype-specific preferences for targeting either template or coding strand of open reading frames. While some DNA-targeting systems (type I-E and type II systems) prefer the template strand and avoid mRNA, other DNA- and RNA-targeting systems (types I-A and I-B and type III systems) prefer the coding strand and mRNA. In addition, we find large-scale evidence that both CRISPR-Cas adaptation machinery and CRISPR arrays are shared between different CRISPR-Cas systems. This could lead to simultaneous DNA and RNA targeting of invaders, which may be effective at combating mobile genetic invaders. This study has broad implications for our understanding of how CRISPR-Cas systems work in a wide range of organisms for which only the genome sequence is known.

中文翻译：

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单个和共存 CRISPR-Cas 系统中的 PAM 重复关联和间隔区选择偏好

适应性 CRISPR-Cas 免疫系统将过去入侵者的序列存储为 CRISPR 阵列中的间隔区，从而提供将入侵者与宿主联系起来的直接证据。绘制 CRISPR 间隔区图谱揭示了 CRISPR-Cas 生物学的许多方面，包括原型间隔区相邻基序 (PAM) 等目标要求。然而，迄今为止的研究受到数据库中映射间隔区数量较少的限制。通过使用庞大的宏基因组序列数据库，我们绘制了来自各种微生物的超过 200,000 个独特 CRISPR 间隔区中的约三分之一，并得出了与特定 CRISPR-Cas 亚型相关的超过 200 个独特 PAM 序列的目录。这些 PAM 进一步用于正确分配 CRISPR 阵列的方向，揭示 CRISPR 重复序列的最后一个核苷酸和 PAM 之间的保守模式。我们还可以推断出针对开放阅读框的模板或编码链的 CRISPR-Cas 亚型特异性偏好。虽然一些 DNA 靶向系统（IE 型和 II 型系统）更喜欢模板链并避免 mRNA，但其他 DNA 和 RNA 靶向系统（IA 和 IB 型以及 III 型系统）更喜欢编码链和 mRNA。此外，我们发现大量证据表明 CRISPR-Cas 适应机制和 CRISPR 阵列在不同的 CRISPR-Cas 系统之间共享。这可能导致 DNA 和 RNA 同时针对入侵者，这可能会有效对抗移动的基因入侵者。这项研究对于我们理解 CRISPR-Cas 系统如何在仅知道基因组序列的多种生物体中发挥作用具有广泛的意义。