CRISPR pp 1-21 | Cite as

Investigating CRISPR RNA Biogenesis and Function Using RNA-seq

  • Nadja Heidrich
  • Gaurav Dugar
  • Jörg Vogel
  • Cynthia M. SharmaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1311)


The development of deep sequencing technology has greatly facilitated transcriptome analyses of both prokaryotes and eukaryotes. RNA-sequencing (RNA-seq), which is based on massively parallel sequencing of cDNAs, has been used to annotate transcript boundaries and revealed widespread antisense transcription as well as a wealth of novel noncoding transcripts in many bacteria. Moreover, RNA-seq is nowadays widely used for gene expression profiling and about to replace hybridization-based approaches such as microarrays. RNA-seq has also informed about the biogenesis and function of CRISPR RNAs (crRNAs) of different types of bacterial RNA-based CRISPR-Cas immune systems. Here we describe several studies that employed RNA-seq for crRNA analyses, with a particular focus on a differential RNA-seq (dRNA-seq) approach, which can distinguish between primary and processed transcripts and allows for a genome-wide annotation of transcriptional start sites. This approach helped to identify a new crRNA biogenesis pathway of Type II CRISPR-Cas systems that involves a trans-encoded small RNA, tracrRNA, and the host factor RNase III.

Key words

RNA-sequencing (RNA-seq) Deep sequencing cDNA library Terminator exonuclease Differential RNA-seq (dRNA-seq) PNK Small RNA identification CRISPR-Cas tracrRNA crRNA 



CRISPR work in the Vogel lab is funded by DFG Grant Vo875/7-1 and the Bavarian BioSysNet program. Work in the Sharma lab is supported by the ZINF Young Investigator program at the Research Center for Infectious Diseases (ZINF) in Würzburg, Germany, the Bavarian BioSysNet program, DFG Grant Sh580/1-1 and the Daimler and Benz foundation. GD is supported by the Graduate School for Life Sciences (GSLS) Würzburg.


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Nadja Heidrich
    • 1
  • Gaurav Dugar
    • 2
  • Jörg Vogel
    • 1
  • Cynthia M. Sharma
    • 2
    Email author
  1. 1.Institute for Molecular Infection Biology (IMIB)University of WürzburgWürzburgGermany
  2. 2.Research Centre for Infectious Diseases (ZINF)University of WürzburgWürzburgGermany

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