The adaptation phase of CRISPR-Cas immunity depends on the precise integration of short segments of foreign DNA (spacers) into a specific genomic location within the CRISPR locus by the Cas1-Cas2 integration complex. Although off-target spacer integration outside of canonical CRISPR arrays has been described in vitro, no evidence of non-specific integration activity has been found in vivo. Here, we show that non-canonical off-target integrations can occur within bacterial chromosomes at locations that resemble the native CRISPR locus by characterizing hundreds of off-target integration locations within Escherichia coli. Considering whether such promiscuous Cas1-Cas2 activity could have an evolutionary role through the genesis of neo-CRISPR loci, we combed existing CRISPR databases and available genomes for evidence of off-target integration activity. This search uncovered several putative instances of naturally occurring off-target spacer integration events within the genomes of Yersinia pestis and Sulfolobus islandicus. These results are important in understanding alternative routes to CRISPR array genesis and evolution, as well as in the use of spacer acquisition in technological applications.

中文翻译：

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通过非规范间隔子整合在体内自发产生CRISPR基因座。

CRISPR-Cas免疫的适应阶段取决于Cas1-Cas2整合复合物能否将短片段外源DNA（间隔子）精确整合到CRISPR基因座内的特定基因组位置。尽管体外已经描述了规范CRISPR阵列之外的脱靶间隔子整合，但尚未在体内发现非特异性整合活性的证据。在这里，我们通过表征大肠杆菌内数百个脱靶整合位点的特征，显示出非经典的脱靶整合位点可以在细菌染色体内与天然CRISPR基因座相似的位置发生。考虑到这种混杂的Cas1-Cas2活性是否可以通过neo-CRISPR基因座的产生而具有进化作用，我们对现有的CRISPR数据库和可用的基因组进行了梳理，以证明脱靶整合活性。这项搜索发现了鼠疫耶尔森菌和Sulfolobus islandicus基因组内自然发生的脱靶间隔子整合事件的几个推定实例。这些结果对于理解CRISPR阵列发生和进化的替代途径以及在技术应用中使用间隔子采集非常重要。