Molina-Sánchez María Dolores, Martínez-Abarca Francisco, Millán Vicenta, Mestre Mario Rodríguez, Stehantsev Pavlo, Stetsenko Artem, Guskov Albert, Toro Nicolás
Department of Soil and Plant Microbiology, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Structure, Dynamics and Function of Bacterial Genomes, Grupo de Ecología Genética de la Rizosfera, C/Profesor Albareda 1, 18008 Granada, Spain.
Section of Microbiology, Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark.
Nucleic Acids Res. 2024 Dec 11;52(22):14229-14243. doi: 10.1093/nar/gkae1154.
Cas13-containing type VI CRISPR-Cas systems specifically target RNA; however, the mechanism of spacer acquisition remains unclear. We have previously reported the association of reverse transcriptase-Cas1 (RT-Cas1) fusion proteins with certain types of VI-A systems. Here, we show that RT-Cas1 fusion proteins are also recruited by type VI-B systems in bacteria from gut microbiomes, constituting a VI-B1 variant system that includes a CorA-encoding locus in addition to the CRISPR array and the RT-Cas1/Cas2 adaptation module. We found that type VI RT-CRISPR systems were functional for spacer acquisition, CRISPR array processing and interference activity, demonstrating that adaptive immunity mediated by these systems can function independently of other in trans systems. We provide evidence that the RT associated with these systems enables spacer acquisition from RNA molecules. We also found that CorA encoded by type VI-B1 RT-associated systems can transport divalent metal ions and downregulate Cas13b-mediated RNA interference. These findings highlight the importance of RTs in RNA-targeting CRISPR-Cas systems, potentially enabling the integration of RNA-derived spacers into CRISPR arrays as a mechanism against RNA-based invaders in specific environments.
含Cas13的VI型CRISPR-Cas系统特异性靶向RNA；然而，间隔序列获取的机制仍不清楚。我们之前报道了逆转录酶-Cas1（RT-Cas1）融合蛋白与某些类型的VI-A系统有关联。在此，我们表明RT-Cas1融合蛋白也被来自肠道微生物群的细菌中的VI-B系统招募，构成了一个VI-B1变体系统，除了CRISPR阵列和RT-Cas1/Cas2适应模块外，该系统还包括一个编码CorA的基因座。我们发现VI型RT-CRISPR系统在间隔序列获取、CRISPR阵列加工和干扰活性方面具有功能，这表明由这些系统介导的适应性免疫可以独立于其他反式系统发挥作用。我们提供证据表明，与这些系统相关的RT能够从RNA分子中获取间隔序列。我们还发现，VI-B1 RT相关系统编码的CorA可以转运二价金属离子并下调Cas13b介导的RNA干扰。这些发现突出了RT在靶向RNA的CRISPR-Cas系统中的重要性，这可能使RNA衍生的间隔序列整合到CRISPR阵列中，作为在特定环境中对抗基于RNA的入侵者的一种机制。
