| Abstract [eng] |
Recently, an adaptive prokaryotic immune system, CRISPR/Cas, was identified that provides acquired immunity against viruses and plasmids. Despite of active research in this field, the molecular mechanism of CRISPR/Cas systems remains not clear. The aims of this study were, to perform genetic and biochemical analyses of the S. thermophilus Type II CRISPR3/Cas system cloned and expressed in the heterologous E. coli host and to determine the mechanism of DNA interference in Type II CRISPR/Cas systems. In this work was demonstrated that CRISPR/Cas system can be transferred across distant genera and provides heterologous interference against invasive nucleic acids, so bacteria might be “vaccinated” against viruses and plasmids in this way. It was established that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, in this work the Cas9–crRNA complex of the S. thermophilus CRISPR3/Cas system has been isolated and demonstrated that it cleaves DNA bearing a nucleotide sequence complementary to the crRNA, in a proto-spacer adjacent motif dependent manner. Sequence specificity of the Cas9–crRNA complex is dictated by the 42-nt crRNA. We showed that DNA cleavage is executed by two distinct active sites (RuvC and HNH) within Cas9 to generate site-specific nicks on opposite DNA strands. By altering the RNA sequence within the Cas9 crRNA complex, programmable endonucleases can be designed both for in vitro and in vivo applications, and this thesis provide a proof of concept for this novel and promising application. |
