CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus.
CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus.
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DOI：
10.1093/nar/gku161
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发表时间：
2014-04
影响因子：
14.9
通讯作者：
Schleper C
中科院分区：
文献类型：
--
作者：
Zebec Z;Manica A;Zhang J;White MF;Schleper C
最近发现的簇状规则间隔短回文重复(CRISPR)介导的病毒防御代表了许多细菌和古菌的适应性免疫系统。小的CRISPR RNA导致互补的入侵核酸与相关蛋白质或蛋白质复合体一起切割。在这里，我们展示了CRISPR介导的切割入侵病毒在极端嗜热的古细菌Sulfolobus solfararicus中的mRNA。定量聚合酶链式反应表明，靶向mRNA有40%以上被切割。Northern分析显示了该基因的切割情况，并绘制了切割位点图。在体外，相同的底物被从Sulfolobus solfararicus纯化的CRISPR相关的CMR复合体切割。体内系统也被重新编程，使用一个人工的微型CRISPR基因座敲除选定的染色体基因(β-半乳糖苷酶)的mRNAs。使用单个互补间隔区，∼可将靶基因和相应的细胞内蛋白活性降低50%。我们的结果表明，在原核生物中，小RNA介导的mRNA在体内被切割(即类似于真核生物中的RNA干扰)，并重新编程以沉默特定的感兴趣基因。
The recently discovered clustered regularly interspaced short palindromic repeat (CRISPR)-mediated virus defense represents an adaptive immune system in many bacteria and archaea. Small CRISPR RNAs cause cleavage of complementary invading nucleic acids in conjunction with an associated protein or a protein complex. Here, we show CRISPR-mediated cleavage of mRNA from an invading virus in the hyperthermophilic archaeon Sulfolobus solfataricus. More than 40% of the targeted mRNA could be cleaved, as demonstrated by quantitative polymerase chain reaction. Cleavage of the mRNA was visualized by northern analyses and cleavage sites were mapped. In vitro, the same substrates were cleaved by the purified CRISPR-associated CMR complex from Sulfolobus solfataricus. The in vivo system was also re-programmed to knock down mRNA of a selected chromosomal gene (β-galactosidase) using an artificial miniCRISPR locus. With a single complementary spacer, ∼50% reduction of the targeted mRNA and of corresponding intracellular protein activity was achieved. Our results demonstrate in vivo cleavage of mRNA in a prokaryote mediated by small RNAs (i.e. analogous to RNA interference in eukaryotes) and the re-programming of the system to silence specific genes of interest.
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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.
影响因子：
14.9
作者：
Berkner S;Grogan D;Albers SV;Lipps G
通讯作者：
Lipps G
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
3.6
作者：
Lillestol, Reidun K.;Shah, Shiraz A.;Garrett, Roger A.
通讯作者：
Garrett, Roger A.
通过反式编码的小 RNA 和宿主因子 RNase III 进行 CRISPR RNA 成熟。
影响因子：
64.8
作者：
Deltcheva, Elitza;Chylinski, Krzysztof;Sharma, Cynthia M.;Gonzales, Karine;Chao, Yanjie;Pirzada, Zaid A.;Eckert, Maria R.;Vogel, Joerg;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle