title: CRISPR transcript processing: a mechanism for generating a large number of small interfering RNAs.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Belgrade, Serbia. dmarko@bio.bg.ac.rs
authors: Marko
authors: M
authors: Djordjevic
authors: Magdalena
authors: M
authors: Djordjevic
authors: Konstantin
authors: K
authors: Severinov
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005784:Gene Amplification; D015964:Gene Expression Regulation, Bacterial; D055029:Inverted Repeat Sequences; D008957:Models, Genetic; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism
chemical_list: D012329:RNA, Bacterial; D034741:RNA, Small Interfering