The prokaryotic adaptive immune system CRISPR/Cas serves as a defense against bacteriophage and invasive nucleic acids. A type I-E CRISPR/Cas system has been detected in classical biotype isolates of Vibrio cholerae, the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5′-TT-3′ protospacer-adjacent motif (PAM) for interference. However, several designed spacers against the 5′-TT-3′ PAM do not interfere as expected, indicating that further investigation of this system is necessary. In this study, we identified additional conserved sequences, including a pyrimidine in the 5′ position of the spacer and a purine in the complementary position of the protospacer using 873 unique spacers and 2,267 protospacers mined from CRISPR arrays in deposited sequences of V. cholerae. We present bioinformatic evidence that during acquisition the protospacer purine is captured in the prespacer and that a 5′-RTT-3′ PAM is necessary for spacer acquisition. Finally, we demonstrate experimentally, by designing and manipulating spacer and cognate PAMs in a plasmid conjugation assay, that a 5′-RTT-3′ PAM is necessary for CRISPR interference, and we discover functional consequences for spacer efficacy related to the identity of the 5′ spacer pyrimidine.

中文翻译：

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霍乱弧菌 IE 型 CRISPR/Cas 系统中间隔区和原型间隔区序列要求的鉴定


原核适应性免疫系统 CRISPR/Cas 可防御噬菌体和侵入性核酸。在霍乱弧菌（霍乱疾病的病原体）的经典生物型分离株中检测到了 IE 型 CRISPR/Cas 系统。该系统的实验表征揭示了一个功能性免疫系统，该系统使用 5'-TT-3' 原型间隔子相邻基序 (PAM) 进行干扰。然而，针对 5'-TT-3' PAM 设计的几种间隔物并未按预期产生干扰，表明有必要对该系统进行进一步研究。在这项研究中，我们使用从霍乱弧菌保藏序列中的 CRISPR 阵列中挖掘的 873 个独特间隔区和 2,267 个原型间隔区，鉴定了其他保守序列，包括间隔区 5' 位置的嘧啶和原型间隔区互补位置的嘌呤。我们提供的生物信息证据表明，在采集过程中，原型间隔区嘌呤被捕获在预间隔区中，并且 5'-RTT-3' PAM 对于间隔区获取是必需的。最后，我们通过在质粒缀合测定中设计和操作间隔区和同源 PAM，通过实验证明 5'-RTT-3' PAM 对于 CRISPR 干扰是必需的，并且我们发现了与间隔区功效相关的功能后果。 5'间隔基嘧啶。