Investigating CRISPR RNA Biogenesis and Function Using RNA-Seq

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 researchers from the University of Würzburg 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.

rna-seqSchematic representation of different RNA sequencing approaches including TEX and PNK treatment and combinations of both treatments.

Heidrich N, Dugar G, Vogel J, Sharma CM. (2015) Investigating CRISPR RNA Biogenesis and Function Using RNA-seq. Methods Mol Biol 1311:1-21. [abstract]

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