Bacterial Type IV CRISPR-Cas systems are thought to rely on multi-subunit ribonucleoprotein complexes to interfere with mobile genetic elements, but the substrate requirements and potential DNA nuclease activities for many systems within this type are uncharacterized. Here we show that the native Pseudomonas oleovorans Type IV-A CRISPR-Cas system targets DNA in a PAM-dependent manner and elicits interference without showing DNA nuclease activity. We found that the first crRNA of P. oleovorans contains a perfect match in the host gene coding for the Type IV pilus biogenesis protein PilN. Deletion of the native Type IV CRISPR array resulted in upregulation of pilN operon transcription in the absence of genome cleavage, indicating that Type IV-A CRISPR-Cas systems can function in host gene regulation. These systems resemble CRISPR interference (CRISPRi) methodology but represent a natural CRISPRi-like system that is found in many Pseudomonas and Klebsiella species and allows for gene silencing using engineered crRNAs.

细菌 IV 型 CRISPR-Cas 系统被认为依赖多亚基核糖核蛋白复合物来干扰移动遗传元件，但这种类型的许多系统的底物要求和潜在的 DNA 核酸酶活性尚不明确。在这里，我们表明天然的Pseudomonas oleovorans IV-A 型 CRISPR-Cas 系统以 PAM 依赖性方式靶向 DNA，并在不显示 DNA 核酸酶活性的情况下引发干扰。我们发现P. oleovorans的第一个 crRNA在编码 IV 型菌毛生物发生蛋白 PilN 的宿主基因中包含完美匹配。天然 IV 型 CRISPR 阵列的删除导致pilN的上调在没有基因组切割的情况下进行操纵子转录，表明 IV-A 型 CRISPR-Cas 系统可以在宿主基因调控中发挥作用。这些系统类似于 CRISPR 干扰 (CRISPRi) 方法，但代表了一种天然的 CRISPRi 样系统，存在于许多假单胞菌和克雷伯菌属物种中，并允许使用工程 crRNA 进行基因沉默。