Data_Sheet_1_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.

在多种莱姆病原体（Leptospira interrogans）血清型中，CRISPR-Cas的亚型I-B位点拥有一个或多个CRISPR阵列。通过预测CRISPR-Cas的在线数据库（CRISPRCasdb），在莱姆病原体血清型Lai中发现了七个CRISPR阵列，这些阵列位于两个独立的cas操纵子之间。在本研究中，我们对血清型Lai的CRISPR亚型I-B位点的重复-间隔边界进行了重新定义。这种对血清型Lai中阵列边界的精细化处理是在与已表征的血清型哥本哈根的阵列以及CRISPR旁侧序列的人工分析之后进行的。利用逆转录-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中的转录本进行的RT-PCR和定量实时PCR分析表明，亚型I-B的七个CRISPR阵列作为一个单一的前体单元进行转录。此外，Cas6对亚型I-B的两个微型前crRNA的切割证明了成熟crRNA的生物发生，这对于指导性干扰外来DNA至关重要。本研究揭示了血清型Lai中转录方向和crRNA生物发生方面的见解，这对于RNA介导的入侵核酸干扰至关重要。