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.

Schematic representation of different RNA sequencing approaches including TEX and PNK treatment and combinations of both treatments.