title: Investigating CRISPR RNA Biogenesis and Function Using RNA-seq.
abstract: 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.
authors.affiliation: Institute for Molecular Infection Biology (IMIB), University of Würzburg, Josef-Schneider-Straße 2/Bau D15, 97080, Würzburg, Germany.
authors.forename: Nadja
authors.initials: N
authors.lastname: Heidrich
authors.forename: Gaurav
authors.initials: G
authors.lastname: Dugar
authors.forename: Jörg
authors.initials: J
authors.lastname: Vogel
authors.forename: Cynthia M
authors.initials: CM
authors.lastname: Sharma
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D005092:Exonucleases / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods*; D014026:Nicotiana / Q000201:enzymology
chemical_list: D018076:DNA, Complementary; D012313:RNA; D005092:Exonucleases; D011755:Pyrophosphatases