Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus
Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus
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DOI：
10.1073/pnas.1905421116
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发表时间：
2019-08-06
影响因子：
11.1
通讯作者：
Barrangou, Rodolphe
中科院分区：
文献类型：
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作者：
Hidalgo-Cantabrana, Claudio;Goh, Yong Jun;Barrangou, Rodolphe
CRISPR-Cas系统现在广泛用于多种生物体中的基因组编辑和转录调控。2类单效应核酸酶（例如Cas9或Cas 12）的紧凑和便携性质促进了植物、动物和微生物中的定向基因组修饰。然而，大多数CRISPR-Cas系统属于更普遍的1类，其取决于多蛋白效应复合物。在本研究中，我们详细介绍了具有5 '-AAA-3'原型间隔区邻近基序（PAM）和61个核苷酸指导CRISPR RNA（crRNA）的天然I-E型CRISPR-Cas系统如何可以重新用于卷曲乳杆菌（Lactobacillus crispatus）中的有效染色体靶向和基因组编辑，卷曲乳杆菌是阴道和肠道中的重要微生物和有益微生物。具体来说，我们产生了不同的突变，包括643个碱基对（bp）缺失（100%的效率），终止密码子插入（36%），和一个单核苷酸取代（19%）的胞外多糖引发糖基转移酶（p-git）。其他遗传靶点包括前噬菌体DNA包装Nut中的308-bp缺失（20%）和烯醇化酶下游绿色荧光蛋白基因的730-bp插入（23%）。这种方法能够灵活地改变以前遗传上不稳定的物种L。卷曲，具有益生菌增强，生物工程和粘膜疫苗递送的潜力。这些结果还为重新利用内源性CRISPR-Cas系统以实现灵活的基因组靶向和编辑提供了框架，同时扩展了工具箱，以包括自然界中发现的最丰富和最多样化的系统之一。
CRISPR-Cas systems are now widely used for genome editing and transcriptional regulation in diverse organisms. The compact and portable nature of class 2 single effector nucleases, such as Cas9 or Cas12, has facilitated directed genome modifications in plants, animals, and microbes. However, most CRISPR-Cas systems belong to the more prevalent class 1 category, which hinges on multi-protein effector complexes. In the present study, we detail how the native type I-E CRISPR-Cas system, with a 5'-AAA-3' protospacer adjacent motif (PAM) and a 61-nucleotide guide CRISPR RNA (crRNA) can be repurposed for efficient chromosomal targeting and genome editing in Lactobacillus crispatus, an important commensal and beneficial microbe in the vaginal and intestinal tracts. Specifically, we generated diverse mutations encompassing a 643-base pair (bp) deletion (100% efficiency), a stop codon insertion (36%), and a single nucleotide substitution (19%) in the exopolysaccharide priming-glycosyl transferase (p-git). Additional genetic targets included a 308-bp deletion (20%) in the prophage DNA packaging Nut and a 730-bp insertion of the green fluorescent protein gene downstream of enolase (23%). This approach enables flexible alteration of the formerly genetically recalcitrant species L. crispatus, with potential for probiotic enhancement, biotherapeutic engineering, and mucosal vaccine delivery. These results also provide a framework for repurposing endogenous CRISPR-Cas systems for flexible genome targeting and editing, while expanding the toolbox to include one of the most abundant and diverse systems found in nature.