Many bacteria and archaea possess an RNA-guided adaptive and inheritable immune system that consists of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. In most CRISPR-Cas systems, the maturation of CRISPR-derived small RNAs (crRNAs) is essential for functionality. Cas6 endonucleases function as the most frequent CRISPR RNA maturation enzymes. In the cyanobacterium Anabaena sp. PCC 7120, ten CRISPR loci are present, but only two cas gene cassettes plus a Tn7-associated eleventh array. In this study, we deleted the two cas6 genes alr1482 (Type III-D) or alr1566 (Type I-D) and tested the specificities of the two corresponding enzymes in the resulting mutant strains, as recombinant proteins and in a cell-free transcription-translation system. The results assign the direct repeats (DRs) to three different groups. While Alr1566 is specific for one group, Alr1482 has a higher preference for the DRs of the second group but can also cleave those of the first group. We found that this cross-recognition limits crRNA accumulation for the Type I-D system in vivo.

We also show that the DR of the cas gene-free CRISPR array of cyanophage N-1 is processed by these enzymes, suggesting that it is fully competent in association with host-encoded Cas proteins. The data support the functionality of CRISPR arrays that frequently appear fragmented to multiple genomic loci in multicellular cyanobacteria and disfavour other possibilities, such as the nonfunctionality of these orphan repeat-spacer arrays. Our results show the functional coordination of Cas6 endonucleases with both neighbouring and remote repeat-spacer arrays in the CRISPR-Cas system of cyanobacteria.

许多细菌和古细菌拥有RNA引导的适应性和可遗传的免疫系统，该系统由成簇的规则间隔的短回文重复序列（CRISPR）和CRISPR相关（Cas）蛋白组成。在大多数CRISPR-Cas系统中，CRISPR衍生的小RNA（crRNA）的成熟对于功能至关重要。Cas6核酸内切酶是最常见的CRISPR RNA成熟酶。在蓝藻鱼腥藻中。PCC 7120，存在十个CRISPR基因座，但是只有两个cas基因盒和一个与Tn7相关的第十一个阵列。在这项研究中，我们删除了两个cas6基因alr1482（III-D型）或alr1566（Type ID），并测试了所得突变株中两种相应酶作为重组蛋白和无细胞转录-翻译系统的特异性。结果将直接重复（DR）分配给三个不同的组。虽然Alr1566是一组特定的，但Alr1482对第二组的DR具有较高的偏好，但也可以切割第一组的DR。我们发现这种交叉识别限制了体内ID类型系统的crRNA积累。

我们还显示了无酶基因的cas CRISPR噬菌体N-1阵列的DR是由这些酶处理的，这表明它与宿主编码的Cas蛋白完全结合。数据支持CRISPR阵列的功能，该阵列经常在多细胞蓝细菌中破碎成多个基因组位点，并不利于其他可能性，例如这些孤儿重复间隔物阵列的无功能性。我们的结果表明，Cas6核酸内切酶与蓝细菌的CRISPR-Cas系统中的相邻和远端重复间隔子阵列均具有功能协调性。