CRISPR(成簇的规则间隔的短回文重复序列)-Cas(CRISPR相关的)系统为原核生物提供了针对外源核酸入侵者的有效保护。我们最近证明了来自艰难梭菌(Clostridium)的CRISPR-Cas系统的防御性干扰功能,一种主要的人类肠病原体,并表明它可以用于这种细菌的有效基因组编辑。然而,对于CRISPR-Cas功能的适应性和序列要求,在这种病原体中干扰和新间隔区的获取仍然缺乏分子细节。尽管在各种原核生物中积累了对单个CRISPR-Cas系统的知识,没有关于细菌I-B型CRISPR-Cas系统适应过程的数据.这里,我们报道了第一个实验证据,即艰难梭菌I-B型CRISPR-Cas系统在其适应模块过表达时获得了新的间隔区。大多数新的间隔区来源于表达适应所需的Cas蛋白的质粒或来源于预期产生游离DNA末端的艰难梭菌基因组区域。来自前间隔区-相邻基序(PAM)文库实验和质粒接合效率测定的结果表明,艰难梭菌CRISPR-Cas需要YCN共有PAM来进行有效干扰。我们揭示了适应和干扰机制之间的功能联系,因为新适应的间隔区来自与CCNPAM相关的序列,这符合干涉共识。本研究中的功能性PAM的定义和多个艰难梭菌CRISPR阵列中的每一个的相对活性水平的建立对于涉及该生物体的进一步基于CRISPR的生物技术和医学应用是必要的。重要性CRISPR-Cas系统为原核生物提供适应性免疫,以防御外来核酸入侵者,如病毒或噬菌体和质粒。CRISPR-Cas系统是高度多样化的,对单个CRISPR-Cas亚型的详细研究对于我们理解微生物适应策略的各个方面以及潜在的应用是重要的。我们工作的意义在于为新兴的人类肠病原体艰难梭菌中的I-B型CRISPR-Cas系统适应提供了第一个实验证据。这种细菌需要在富含噬菌体的肠道群落中生存,及其活跃的CRISPR-Cas系统可能通过获取构成记忆的新间隔区来提供有效的抗噬菌体防御,以进一步消除入侵者。我们的研究还揭示了适应和干扰CRISPR机制之间的功能联系。外源核酸序列中所有可能的功能性三核苷酸基序上游前间隔区的定义对于该病原体中基于CRISPR的基因组编辑和开发针对艰难梭菌感染的新药是重要的。
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems provide prokaryotes with efficient protection against foreign nucleic acid invaders. We have recently demonstrated the defensive interference function of a CRISPR-Cas system from Clostridioides (Clostridium) difficile, a major human enteropathogen, and showed that it could be harnessed for efficient genome editing in this bacterium. However, molecular details are still missing on CRISPR-Cas function for adaptation and sequence requirements for both interference and new spacer acquisition in this pathogen. Despite accumulating knowledge on the individual CRISPR-Cas systems in various prokaryotes, no data are available on the adaptation process in bacterial type I-B CRISPR-Cas systems. Here, we report the first experimental evidence that the C. difficile type I-B CRISPR-Cas system acquires new spacers upon overexpression of its adaptation module. The majority of new spacers are derived from a plasmid expressing Cas proteins required for adaptation or from regions of the C. difficile genome where generation of free DNA termini is expected. Results from protospacer-adjacent motif (PAM) library experiments and plasmid conjugation efficiency assays indicate that C. difficile CRISPR-Cas requires the YCN consensus PAM for efficient interference. We revealed a functional link between the adaptation and interference machineries, since newly adapted spacers are derived from sequences associated with a CCN PAM, which fits the interference consensus. The definition of functional PAMs and establishment of relative activity levels of each of the multiple C. difficile CRISPR arrays in present study are necessary for further CRISPR-based biotechnological and medical applications involving this organism. IMPORTANCE CRISPR-Cas systems provide prokaryotes with adaptive immunity for defense against foreign nucleic acid invaders, such as viruses or phages and plasmids. The CRISPR-Cas systems are highly diverse, and detailed studies of individual CRISPR-Cas subtypes are important for our understanding of various aspects of microbial adaptation strategies and for the potential applications. The significance of our work is in providing the first experimental evidence for type I-B CRISPR-Cas system adaptation in the emerging human enteropathogen Clostridioides difficile. This bacterium needs to survive in phage-rich gut communities, and its active CRISPR-Cas system might provide efficient antiphage defense by acquiring new spacers that constitute memory for further invader elimination. Our study also reveals a functional link between the adaptation and interference CRISPR machineries. The definition of all possible functional trinucleotide motifs upstream protospacers within foreign nucleic acid sequences is important for CRISPR-based genome editing in this pathogen and for developing new drugs against C. difficile infections.