Data_Sheet_2_Transcriptional analysis of CRISPR I-B arrays of Leptospira interrogans serovar Lai and its processing by Cas6.PDF

In the genome of various Leptospira interrogans serovars, the subtype I-B locus of CRISPR-Cas possesses either one or multiple CRISPR arrays. In silico database (CRISPRCasdb) for predicting CRISPR-Cas reveals seven CRISPR arrays in L. interrogans serovar Lai positioned between the two independent cas-operons. Here, we present the redefined repeat-spacer boundaries of the CRISPR subtype I-B locus of serovar Lai. Such refinement of boundaries of arrays in serovar Lai was done after comparison with the characterized array of another serovar Copenhageni and the manual analysis of CRISPR flanking sequences. Using the reverse transcription-PCR (RT-PCR), we account that the seven CRISPR are transcriptionally active in serovar Lai. Our RT-PCR and quantitative real-time PCR analysis of transcripts in serovar Lai indicated that seven CRISPR of subtype I-B transcribe together as a single precursor unit. Moreover, the cleavage of the two miniature pre-crRNA of the subtype I-B by Cas6 demonstrates the biogenesis of the expected size of mature crRNA essential for the guided interference of foreign DNA. This study features insight into transcription direction and the crRNA biogenesis in serovar Lai essential for RNA-mediated interference of invading nucleic acids.

在多种莱姆螺旋体（Leptospira interrogans）血清型中，CRISPR-Cas的I-B亚型基因座含有单个或多个CRISPR阵列。通过对CRISPR-Cas进行在硅数据库（CRISPRCasdb）预测，发现在莱姆螺旋体血清型Lai中存在七个CRISPR阵列，它们位于两个独立的cas操纵子之间。本研究重新定义了莱姆螺旋体血清型Lai中CRISPR I-B亚型重复序列与间隔序列的边界。这种对莱姆螺旋体血清型Lai中阵列边界的细化是在与另一个血清型哥本哈根（Copenhageni）的特征性阵列以及CRISPR旁侧序列的手动分析后进行的。通过逆转录-聚合酶链反应（RT-PCR），我们证实了血清型Lai中的七个CRISPR阵列在转录上具有活性。我们对血清型Lai中转录本的RT-PCR和定量实时PCR分析表明，七个I-B亚型的CRISPR序列作为一个单一的预制单元进行转录。此外，Cas6对I-B亚型两种微型前crRNA的切割表明，成熟的crRNA的生物发生符合指导外源DNA干扰所需的预期大小。本研究揭示了莱姆螺旋体血清型Lai中转录方向和crRNA生物发生的重要性，这对于介导RNA的入侵核酸干扰至关重要。