Aside from providing adaptive immunity, type I CRISPR-Cas was recently unearthed to employ a noncanonical RNA guide (CreA) to transcriptionally repress an RNA toxin (CreT). Here, we report that, for most archaeal and bacterial CreTA modules, the creA gene actually carries two flanking ‘CRISPR repeats’, which are, however, highly divergent and degenerated. By deep sequencing, we show that the two repeats give rise to an 8-nt 5′ handle and a 22-nt 3′ handle, respectively, i.e., the conserved elements of a canonical CRISPR RNA, indicating they both retained critical nucleotides for Cas6 processing during divergent degeneration. We also uncovered a minimal CreT toxin that sequesters the rare transfer RNA for isoleucine, tRNAIleCAU, with a six-codon open reading frame containing two consecutive AUA codons. To fully relieve its toxicity, both tRNAIleCAU overexpression and supply of extra agmatine (modifies the wobble base of tRNAIleCAU to decipher AUA codons) are required. By replacing AUA to AGA/AGG codons, we reprogrammed this toxin to sequester rare arginine tRNAs. These data provide essential information on CreTA origin and for future CreTA prediction, and enrich the knowledge of tRNA-sequestering small RNAs that are employed by CRISPR-Cas to get addictive to the host.

中文翻译：

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来自最小 CRISPR 的 creA 抗毒素基因的发散变性和 tRNA 螯合 CreT 毒素的收敛策略

除了提供适应性免疫外，最近还发现 I 型 CRISPR-Cas 采用非经典 RNA 指导 (CreA) 来转录抑制 RNA 毒素 (CreT)。在这里，我们报告说，对于大多数古细菌和细菌 CreTA 模块，creA 基因实际上带有两个侧翼的“CRISPR 重复”，然而，它们是高度分化和退化的。通过深度测序，我们表明这两个重复分别产生了一个 8-nt 5' 手柄和一个 22-nt 3' 手柄，即典型 CRISPR RNA 的保守元件，表明它们都保留了 Cas6 的关键核苷酸发散退化过程中的处理。我们还发现了一种最小的 CreT 毒素，它用一个包含两个连续 AUA 密码子的六密码子开放阅读框来隔离异亮氨酸的稀有转移 RNA，tRNAIleCAU。为充分解除其毒性，tRNAIleCAU 过表达和额外胍丁胺的供应（修改 tRNAIleCAU 的摆动碱基以破译 AUA 密码子）都是必需的。通过将 AUA 替换为 AGA/AGG 密码子，我们将这种毒素重新编程以隔离稀有的精氨酸 tRNA。这些数据提供了有关 CreTA 起源和未来 CreTA 预测的基本信息，并丰富了 CRISPR-Cas 用于使宿主上瘾的 tRNA 隔离小 RNA 的知识。