Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System
Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System
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DOI：
10.1128/aem.01453-16
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发表时间：
2016-09-01
影响因子：
4.4
通讯作者：
Altenbuchner, Josef
中科院分区：
文献类型：
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作者：
Altenbuchner, Josef
簇状规则间隔短回文重复(CRISPR)相关(Cas)系统是细菌的适应性免疫系统。化脓性链球菌的II型CRISPR-Cas9系统最近已发展成为原核生物和真核生物的基因组工程工具。在这里，我们提出了一种单质粒系统，它可以有效地编辑枯草芽孢杆菌的基因组。PJOE8999是一种穿梭载体，对大肠杆菌具有最小的PUC复制起始点，对枯草杆菌有对温度敏感的复制起始点，在两种生物中都有卡那霉素抗性基因。在基因组编辑方面，它携带有受枯草杆菌甘露糖可诱导启动子P-manP控制的Cas9基因和通过强启动子转录的单引导RNA(SgRNA)编码序列。这种sgRNA引导Cas9核酸酶到达它的靶点。SgRNA序列5‘端的20个核苷酸间隔区序列负责靶标特异性，位于BsaI位点之间。因此，通过在BsaI位点之间配对的寡核苷酸改变间隔区序列来改变靶标的特异性。Cas9与sgRNA形成复合体，在其靶点诱导双链断裂(DSB)。DSB的修复和所需的基因组修改是通过添加同源模板来实现的，通常从目标序列的两侧获得两个PCR片段。两个相邻的SfiI位点使得这些同源模板能够有序整合到载体中。通过在枯草杆菌染色体上引入两个大的缺失和修复枯草杆菌168的trpC2突变，证明了CRISPR-Cas9载体的功能。摘要在原核生物中，大多数用于无疤痕基因组工程的方法是基于选择-反选择系统。缺点通常是缺乏合适的反选择标记，反选择所需化合物的毒性，以及目标菌株需要某些突变。CRISPR-CAS系统是近年来发展起来的基因组编辑的重要工具。为枯草杆菌基因组编辑构建的单质粒系统克服了反选择方法的问题。它允许删除和引入点突变。它易于操作，效率很高，可以改装成用于其他硬质材料。
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system from Streptococcus pyogenes has recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing of Bacillus subtilis. The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication for Escherichia coli, the temperature-sensitive replication origin of plasmid pE194(ts) for B. subtilis, and a kanamycin resistance gene working in both organisms. For genome editing, it carries the cas9 gene under the control of the B. subtilis mannose-inducible promoter P-manP and a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. This sgRNA guides the Cas9 nuclease to its target. The 20-nucleotide spacer sequence at the 5' end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site. Repair of the DSBs and the required modification of the genome are achieved by adding homology templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homology templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in the B. subtilis chromosome and by repair of the trpC2 mutation of B. subtilis 168.IMPORTANCEIn prokaryotes, most methods used for scarless genome engineering are based on selection-counterselection systems. The disadvantages are often the lack of a suitable counterselection marker, the toxicity of the compounds needed for counterselection, and the requirement of certain mutations in the target strain. CRISPR-Cas systems were recently developed as important tools for genome editing. The single-plasmid system constructed for the genome editing of B. subtilis overcomes the problems of counterselection methods. It allows deletions and introduction of point mutations. It is easy to handle and very efficient, and it may be adapted for use in other firmicutes.