Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands.
Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands.
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DOI：
10.1371/journal.pgen.1003454
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发表时间：
2013-04
期刊：
影响因子：
4.5
通讯作者：
Fineran PC
中科院分区：
文献类型：
--
作者：
Vercoe RB;Chang JT;Dy RL;Taylor C;Gristwood T;Clulow JS;Richter C;Przybilski R;Pitman AR;Fineran PC
在原核生物中，聚集规律间隔的短回文重复序列（crispr）及其相关（Cas）蛋白构成了抵御噬菌体和质粒的防御系统。CRISPR/Cas系统从外源遗传元素中获取短间隔序列，并将其整合到CRISPR阵列中，从而产生对过去入侵者的记忆。防御是由短的非编码rna提供的，它引导Cas蛋白切割互补的核酸。虽然大多数间隔物是从噬菌体和质粒中获得的，但也有间隔物与宿主细菌染色体中其他地方的基因相匹配的例子。在Pectobacterium atrosepticum中，I-F型CRISPR/Cas系统获得了一个自我互补的间隔子，该间隔子与参与植物致病性的水平获得岛（HAI2）中的原间隔子靶点完美匹配。鉴于缺乏关于CRISPR/ cas介导的染色体靶向的实验数据，我们通过开发一个严格控制的系统来检查这一过程。染色体靶向通过靶向DNA产生高毒性，导致生长抑制和细胞丝化。通过cas操纵子、CRISPR重复序列、protospacer靶点和靶点旁边的protospacer邻近基序（PAM）的突变，避免了毒性表型。事实上，天然的自靶向间隔物是无毒的，因为在PAM序列中，靶点附近有一个核苷酸突变。此外，我们表明染色体靶向可以导致大规模的基因组改变，包括整个先前存在的致病性岛的重塑或删除。这些特征可以被设计用于细菌染色体大区域的靶向删除。总之，在DNA靶向CRISPR/Cas系统中，染色体干扰是有害的，它会导致DNA损伤，并为基因组改变提供强大的选择压力，这可能对细菌进化和致病性产生影响。细菌已经进化出了保护自己免受病毒和其他外来元素持续入侵的机制。被称为CRISPR/Cas的抗性系统最近被发现，并为细菌和古细菌提供了“适应性免疫系统”。这种适应性免疫提供了一种高度可进化的序列特异性小rna记忆，用于记忆过去病毒和外来遗传元素的入侵。在许多情况下，这些系统似乎针对细菌宿主自身基因组内的区域（可能是自身免疫），但其进化原理尚不清楚。在这里，我们证明CRISPR/Cas靶向宿主染色体是高毒性的，但细胞通过减轻免疫机制的突变存活。我们利用这种表型来深入了解这些系统的功能，并表明细菌基因组可能发生巨大变化。例如，针对染色体致病性岛（对马铃薯病原体atrosepticum Pectobacterium的毒力很重要），导致占细菌基因组约2%的岛缺失。这些结果对于CRISPR/Cas系统的作用及其对细菌基因组进化和毒力的影响具有广泛的意义。此外，本研究还证明了它们作为细菌染色体特定区域靶向删除工具的潜力。
In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas–mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA–targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity. Bacteria have evolved mechanisms that provide protection from continual invasion by viruses and other foreign elements. Resistance systems, known as CRISPR/Cas, were recently discovered and equip bacteria and archaea with an “adaptive immune system.” This adaptive immunity provides a highly evolvable sequence-specific small RNA–based memory of past invasions by viruses and foreign genetic elements. There are many cases where these systems appear to target regions within the bacterial host's own genome (a possible autoimmunity), but the evolutionary rationale for this is unclear. Here, we demonstrate that CRISPR/Cas targeting of the host chromosome is highly toxic but that cells survive through mutations that alleviate the immune mechanism. We have used this phenotype to gain insight into how these systems function and show that large changes in the bacterial genome can occur. For example, targeting of a chromosomal pathogenicity island, important for virulence of the potato pathogen Pectobacterium atrosepticum, resulted in deletion of the island, which constituted ∼2% of the bacterial genome. These results have broad significance for the role of CRISPR/Cas systems and their impact on the evolution of bacterial genomes and virulence. In addition, this study demonstrates their potential as a tool for the targeted deletion of specific regions of bacterial chromosomes.
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影响因子：
16
作者：
Hale, Caryn R.;Majumdar, Sonali;Elmore, Joshua;Pfister, Neil;Compton, Mark;Olson, Sara;Resch, Alissa M.;Glover, Claiborne V. C., III;Graveley, Brenton R.;Terns, Rebecca M.;Terns, Michael P.
通讯作者：
Terns, Michael P.
DOI：
10.1007/bf00339621
发表时间：
1988-08-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
CAILLETFAUQUET, P;MAENHAUTMICHEL, G
通讯作者：
MAENHAUTMICHEL, G
影响因子：
5.5
作者：
Brodt A;Lurie-Weinberger MN;Gophna U
通讯作者：
Gophna U
影响因子：
3.2
作者：
Deveau, Helene;Barrangou, Rodolphe;Moineau, Sylvain
通讯作者：
Moineau, Sylvain
影响因子：
4.5
作者：
Hale, Caryn;Kleppe, Kyle;Terns, Michael P.
通讯作者：
Terns, Michael P.