Institute of Molecular Genetics of the Russian Academy of Sciences, Moscow, Russia.
RNA Biol. 2013 May;10(5):716-25. doi: 10.4161/rna.24325. Epub 2013 Apr 25.
In Escherichia coli, the acquisition of new CRISPR spacers is strongly stimulated by a priming interaction between a spacer in CRISPR RNA and a protospacer in foreign DNA. Priming also leads to a pronounced bias in DNA strand from which new spacers are selected. Here, ca. 200,000 spacers acquired during E. coli type I-E CRISPR/Cas-driven plasmid elimination were analyzed. Analysis of positions of plasmid protospacers from which newly acquired spacers have been derived is inconsistent with spacer acquisition machinery sliding along the target DNA as the primary mechanism responsible for strand bias during primed spacer acquisition. Most protospacers that served as donors of newly acquired spacers during primed spacer acquisition had an AAG protospacer adjacent motif, PAM. Yet, the introduction of multiple AAG sequences in the target DNA had no effect on the choice of protospacers used for adaptation, which again is inconsistent with the sliding mechanism. Despite a strong preference for an AAG PAM during CRISPR adaptation, the AAG (and CTT) triplets do not appear to be avoided in known E. coli phages. Likewise, PAM sequences are not avoided in Streptococcus thermophilus phages, indicating that CRISPR/Cas systems may not have been a strong factor in shaping host-virus interactions.
在大肠杆菌中，CRISPR RNA 中的一个间隔序列与外源 DNA 中的一个原间隔序列之间的引发相互作用强烈刺激了新的 CRISPR 间隔序列的获取。引发作用还导致新间隔序列的选择存在明显的 DNA 链偏向性。在这里，分析了大肠杆菌 I-E 型 CRISPR/Cas 驱动的质粒消除过程中获得的大约 20 万个间隔序列。对新获得的间隔序列所源自的质粒原间隔序列位置的分析结果与间隔序列获取机制沿着靶 DNA 滑动不一致，后者是引发时获得的有偏向性间隔序列的主要机制。在引发时获得新间隔序列的大多数原间隔序列都具有 AAG 原间隔邻近基序 (PAM)。然而，在靶 DNA 中引入多个 AAG 序列对用于适应的原间隔序列的选择没有影响，这再次与滑动机制不一致。尽管在 CRISPR 适应过程中强烈偏好 AAG PAM，但在已知的大肠杆菌噬菌体中，AAG（和 CTT）三核苷酸似乎并未被避免。同样，在嗜热链球菌噬菌体中也未避免 PAM 序列，表明 CRISPR/Cas 系统可能不是影响宿主-病毒相互作用的重要因素。
