Type III CRISPR complexes from Thermus thermophilus.

Marta Szychowska; Wojciech Siwek; Damian Pawolski; Asgar Abbas Kazrani; Krzysztof Pyrc; Matthias Bochtler

doi:10.18388/abp.2016_1261

Marta Szychowska International Institute of Molecular and Cell Biology, Warsaw, Poland; Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Łódź, Poland.;
Wojciech Siwek International Institute of Molecular and Cell Biology, Warsaw, Poland.;
Damian Pawolski International Institute of Molecular and Cell Biology, Warsaw, Poland.;
Asgar Abbas Kazrani International Institute of Molecular and Cell Biology, Warsaw, Poland.;
Krzysztof Pyrc Microbiology Department, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; Malopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland.;
Matthias Bochtler International Institute of Molecular and Cell Biology, Warsaw, Poland; Institute of Biochemistry and Biophysics PAS, Warsaw, Poland.;

DOI: https://doi.org/10.18388/abp.2016_1261

Abstract

Pathogen-specific acquired immunity in bacteria is mediated by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems. Thermus thermophilus strain HB8 contains CRISPR systems of several major subtypes (type I, IIIA and IIIB), and has become a widely studied model for CRISPR biology. We have selected two highly expressed CRISPR spacers, crRNA 2.1 and crRNA 2.2, and have enriched endogenous T. thermophilus proteins that co-purify with these crRNAs. Mass spectroscopy indicates that the chromatography protocol enriches predominantly Csm complex subunits, but also Cmr subunits. After several chromatographic steps, size exclusion chromatography indicated a molecular mass of the crRNA associated complex of 265±69 kDa. In agreement with earlier work, crRNAs of different lengths (containing the selected spacers) were observed. Most of these were completely lost when several T. thermophilus csm genes were ablated.