CRISPR transcript processing: a mechanism for generating a large number of small interfering RNAs.
CRISPR transcript processing: a mechanism for generating a large number of small interfering RNAs.
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DOI：
10.1186/1745-6150-7-24
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发表时间：
2012-07-31
期刊：
影响因子：
5.5
通讯作者：
Severinov K
中科院分区：
文献类型：
--
作者：
Djordjevic M;Djordjevic M;Severinov K
CRISPR/Cas是新近发现的一种针对外源DNA的原核防御系统，包括病毒和质粒。CRISPR盒转录为一个连续的转录本(前crRNA)，它被Cas蛋白质加工成负责防御入侵病毒的小RNA分子(CrRNAs)。在大肠杆菌中的实验报告说，cas基因的过度表达只从少数前crRNA产生了大量的crRNA。在这里，我们开发了一个CRISPR处理的最小模型，我们基于现有的实验数据将其参数化。从模型中，我们表明，系统可以生成大量的crRNAs，而只需少量减少前crRNAs的数量。前crRNAs的减少与crRNAs的增加对应着强线性扩增。有趣的是，这种强大的扩增关键依赖于一种未知核酸酶对前crRNA的快速非特异性降解。我们发现，超过一定水平的CaS基因的过度表达并不会导致crRNA的进一步增加，但如果提高CRISPR的转录速率，这种饱和可以得到缓解。我们进一步表明，CRISPR转录速率的小幅增加可以显著降低获得所需数量的crRNA所需的cas基因激活的程度。这里开发的简单数学模型能够解释现有的在大肠杆菌中处理CRISPR转录的实验观察。该模型表明，特定的前crRNA加工和非特异性降解之间的竞争决定了crRNA的稳定水平，并导致了当cas基因过度表达时crRNAs的强线性扩增。该模型进一步说明了细胞中正常存在的几个前crRNA分子的消失如何导致cas过表达时crRNA的大幅增加(两个数量级)。这种大幅增加的一个关键因素是一种未指定的核酸酶的快速非特异性降解，这表明一种尚未确定的核酸酶(S)是CRISPR反应的主要控制元件。转录调控可能是另一个重要的调控机制，因为它既可以增加前crRNA的生成量，也可以改变cas基因的活性水平。本文由米哈伊尔·盖尔芬德、尤金·库宁和L·阿拉文德撰写。
CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated sequences) is a recently discovered prokaryotic defense system against foreign DNA, including viruses and plasmids. CRISPR cassette is transcribed as a continuous transcript (pre-crRNA), which is processed by Cas proteins into small RNA molecules (crRNAs) that are responsible for defense against invading viruses. Experiments in E. coli report that overexpression of cas genes generates a large number of crRNAs, from only few pre-crRNAs. We here develop a minimal model of CRISPR processing, which we parameterize based on available experimental data. From the model, we show that the system can generate a large amount of crRNAs, based on only a small decrease in the amount of pre-crRNAs. The relationship between the decrease of pre-crRNAs and the increase of crRNAs corresponds to strong linear amplification. Interestingly, this strong amplification crucially depends on fast non-specific degradation of pre-crRNA by an unidentified nuclease. We show that overexpression of cas genes above a certain level does not result in further increase of crRNA, but that this saturation can be relieved if the rate of CRISPR transcription is increased. We furthermore show that a small increase of CRISPR transcription rate can substantially decrease the extent of cas gene activation necessary to achieve a desired amount of crRNA. The simple mathematical model developed here is able to explain existing experimental observations on CRISPR transcript processing in Escherichia coli. The model shows that a competition between specific pre-crRNA processing and non-specific degradation determines the steady-state levels of crRNA and is responsible for strong linear amplification of crRNAs when cas genes are overexpressed. The model further shows how disappearance of only a few pre-crRNA molecules normally present in the cell can lead to a large (two orders of magnitude) increase of crRNAs upon cas overexpression. A crucial ingredient of this large increase is fast non-specific degradation by an unspecified nuclease, which suggests that a yet unidentified nuclease(s) is a major control element of CRISPR response. Transcriptional regulation may be another important control mechanism, as it can either increase the amount of generated pre-crRNA, or alter the level of cas gene activity. This article was reviewed by Mikhail Gelfand, Eugene Koonin and L Aravind.
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影响因子：
2.8
作者：
Pourcel, C;Salvignol, G;Vergnaud, G
通讯作者：
Vergnaud, G
影响因子：
3.9
作者：
Mojica, FJM;Díez-Villaseñor, C;Soria, E
通讯作者：
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DOI：
10.1126/science.1159689
发表时间：
2008-08-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
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通讯作者：
van der Oost J
影响因子：
2.8
作者：
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通讯作者：
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影响因子：
5.5
作者：
Makarova, Kira S.;Grishin, Nick V.;Koonin, Eugene V.
通讯作者：
Koonin, Eugene V.