Our research group investigates RNA processing mechanisms that allow us to obtain insights into the coevolution of prokaryotes (Bacteria and Archaea) and viruses. The two main areas of research are (i) the fragmentation of transfer RNA genes and (ii) the function of CRISPR/Cas systems. CRISPR/Cas represents adaptive immune systems that mediate the defense against viruses and conjugative plasmids in the genomes of many bacteria and nearly all archaea. CRISPR elements are characterized by repeat elements that are interspersed by spacer sequences that are often fragments of viral genomes. New spacers can be incorporated into a growing CRISPR cluster. The CRISPR array is transcribed and processed within the repeats, which results in the generation of small CRISPR RNAs (crRNAs). These crRNAs can detect viral DNA during a repeat attack of a virus via base complementarity and commence the destruction of the foreign DNA. Several Cas proteins fulfill the main tasks of a CRISPR system: (i) the acquisition of new spacers in a growing CRISPR array, (ii) the processing of crRNAs and (iii) the recognition and destruction of foreign DNA.
The main goal of this project is to understand the influence of designed spacer sequences on the processing pattern of crRNAs. Therefore, a minimal CRISPR/Cas I-B system will be synthesized which facilitates the directed introduction of artificial spacer units. Such CRISPR/Cas modules can then be utilized to direct the defense against foreign DNA in synthetic bacterial or archaeal cells. RNA-Seq analyses revealed that individual spacer sequences can have drastic effects on CRISPR array transcription and the abundance of crRNAs. These influences will be analyzed in a controlled Escherichia coli system to allow the validation of the efficiency of synthetic CRISPR/Cas modules and accordingly to guarantee protection against foreign DNA.