Abstract：
CRISPR-Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency of most systems against bona fide targets. As such, most primed adaptation to date has been studied within the context of imperfect sequence complementarity between spacers and targets. Here, we take advantage of a native type I-C CRISPR-Cas system in Legionella pneumophila that displays robust primed adaptation even within the context of a perfectly matched target. Using next-generation sequencing to survey acquired spacers, we observe strand bias and positional preference that are consistent with a 3\'-5\' translocation of the adaptation machinery. We show that spacer acquisition happens in a wide range of frequencies across the plasmid, including a remarkable hotspot that predominates irrespective of the priming strand. We systematically characterize protospacer sequence constraints in both adaptation and interference and reveal extensive flexibilities regarding the protospacer adjacent motif in both processes. Lastly, in a strain with a genetically truncated CRISPR array, we observe increased interference efficiency, which, when coupled with forced maintenance of a targeted plasmid, provides a useful experimental system to study spacer loss. Based on these observations, we propose that the Legionella pneumophila type I-C system represents a powerful model to study primed adaptation and the interplay between CRISPR interference and adaptation.
摘要：
CRISPR-Cas是一种细菌和古细菌的适应性免疫系统，入侵者衍生的序列称为间隔区，以靶向侵入性核酸。在认识到以前遇到的入侵者后，该系统可以刺激二次间隔物的收购，称为启动适应的过程。由于大多数系统对真正目标的固有高干扰效率，因此先前对启动自适应的研究变得复杂。因此，迄今为止，已经在间隔区和靶标之间的不完全序列互补性的背景下研究了大多数启动的适应。这里,我们在嗜肺军团菌中利用了天然I-C型CRISPR-Cas系统,该系统即使在完全匹配的目标范围内也表现出强大的启动适应性.使用下一代测序来调查获得的间隔区，我们观察到的股偏倚和位置偏好与适应机制的3'-5'易位一致。我们证明间隔子的获取发生在整个质粒的宽频率范围内，包括一个显著的热点，不管引发链如何。我们系统地表征了原型间隔区序列在适应和干扰方面的约束，并揭示了两个过程中关于原型间隔区相邻基序的广泛灵活性。最后,在具有基因截短的CRISPR阵列的菌株中，我们观察到干扰效率增加，which,当与靶向质粒的强制维持相结合时，提供了一个有用的实验系统来研究间隔损失。基于这些观察,我们认为嗜肺军团菌I-C型系统代表了一个强大的模型来研究启动适应以及CRISPR干扰和适应之间的相互作用。
