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Abstract：
CRISPR DNA arrays of unique spacers separated by identical repeats ensure prokaryotic immunity through specific targeting of foreign nucleic acids complementary to spacers. New spacers are acquired into a CRISPR array in a process of CRISPR adaptation. Selection of foreign DNA fragments to be integrated into CRISPR arrays relies on PAM (protospacer adjacent motif) recognition, as only those spacers will be functional against invaders. However, acquisition of different PAM-associated spacers proceeds with markedly different efficiency from the same DNA. Here, we used a combination of bioinformatics and experimental approaches to understand factors affecting the efficiency of acquisition of spacers by the Escherichia coli type I-E CRISPR-Cas system, for which two modes of CRISPR adaptation have been described: naive and primed. We found that during primed adaptation, efficiency of spacer acquisition is strongly negatively affected by the presence of an AAG trinucleotide-a consensus PAM-within the sequence being selected. No such trend is observed during naive adaptation. The results are consistent with a unidirectional spacer selection process during primed adaptation and provide a specific signature for identification of spacers acquired through primed adaptation in natural populations.IMPORTANCE Adaptive immunity of prokaryotes depends on acquisition of foreign DNA fragments into CRISPR arrays as spacers followed by destruction of foreign DNA by CRISPR interference machinery. Different fragments are acquired into CRISPR arrays with widely different efficiencies, but the factors responsible are not known. We analyzed the frequency of spacers acquired during primed adaptation in an E. coli CRISPR array and found that AAG motif was depleted from highly acquired spacers. AAG is also a consensus protospacer adjacent motif (PAM) that must be present upstream from the target of the CRISPR spacer for its efficient destruction by the interference machinery. These results are important because they provide new information on the mechanism of primed spacer acquisition. They add to other previous evidence in the field that pointed out to a \"directionality\" in the capture of new spacers. Our data strongly suggest that the recognition of an AAG PAM by the interference machinery components prior to spacer capture occludes downstream AAG sequences, thus preventing their recognition by the adaptation machinery.
摘要：
由相同重复序列分开的独特间隔区的CRISPRDNA阵列通过特异性靶向与间隔区互补的外源核酸来确保原核免疫。在CRISPR适应过程中获得新的间隔区进入CRISPR阵列。选择整合到CRISPR阵列中的外源DNA片段依赖于PAM（原型间隔区相邻基序）识别，因为只有那些间隔物才对入侵者起作用。然而,从同一DNA获得不同的PAM相关间隔区的效率明显不同。这里,我们使用生物信息学和实验方法相结合的方法来了解影响大肠杆菌I-E型CRISPR-Cas系统获得间隔区效率的因素，已经描述了CRISPR适应的两种模式：幼稚和预备。我们发现在启动适应过程中，所选择的序列中AAG三核苷酸-共有PAM的存在对间隔区获取的效率产生强烈的负面影响。在幼稚适应期间没有观察到这种趋势。结果与引发适应过程中的单向间隔选择过程一致，并为鉴定通过自然种群中的引发适应获得的间隔区提供了特定的特征。重要性原核生物的适应性免疫取决于将外源DNA片段获取到CRISPR阵列中作为间隔区，然后通过CRISPR干扰机制破坏外源DNA。不同的片段以广泛不同的效率获得到CRISPR阵列中，但原因尚不清楚。我们分析了在大肠杆菌CRISPR阵列中引发的适应过程中获得的间隔区的频率，发现AAG基序从高度获得的间隔区耗尽。AAG也是共识的前间隔区相邻基序（PAM），必须存在于CRISPR间隔区靶标的上游，以便通过干扰机制有效破坏。这些结果很重要，因为它们提供了有关引发的间隔物获取机制的新信息。他们增加了该领域的其他先前证据，这些证据指出了捕获新间隔物的“方向性”。我们的数据强烈表明，在间隔区捕获之前，干扰机械成分对AAGPAM的识别会阻塞下游AAG序列，从而阻止了适应机制对它们的认可。
