Structural and biochemical characterization of the CRISPR-Lon/T complex

• Applicant: Privatdozent Dr. Gregor Hagelueken
• Subject Area: Structural Biology ; Biochemistry
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 504766164

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an adaptive immune system found in many bacteria and archaea. It stores “memories” of past viral attacks in the genome of the host bacterium, in the form of short sections of viral nucleic acids (30-40 base pairs). These “memories” are transcribed and incorporated into RNA-protein sensor complexes. In the event of a renewed attack, these sensor complexes can detect and destroy the corresponding foreign nucleic acids. A large number of different CRISPR systems have been discovered and investigated in recent years. The type III CRISPR systems turned out to be particularly sophisticated. Here, the sensor complex synthesizes so-called cyclic oligonucleotides, when it detects viral nucleic acids. These signal molecules activate effector proteins, which then trigger a complex response to the viral attack. To date, mainly effector proteins with nuclease activities have been studied. These target the host's own nucleic acids and thereby put the host cell into a dormant state. In this state, the cell can better withstand the viral attack. The content of this application is the investigation of a newly discovered type III CRISPR effector called CRISPR-Lon. Our preliminary work revealed that this 60 kDa protein forms a functional unit with the smaller (34 kDa) CRISPR-T protein. In contrast to the mentioned effector nucleases, CRISPR Lon is a protease, which is activated by a cyclic oligonucleotide with 4 AMP subunits. The protease cuts and presumably activates CRISPR-T, which appears to be a sequence-specific ribonuclease.The CRISPR-Lon / -T system shows a completely new aspect of type III CRISPR systems and has astonishing similarities to eukaryotic innate immunity, where proteases such as caspases are used to quickly respond to “emergency” situations. The experiments in this application are designed to fully characterize the CRISPR-Lon / -T system and to answer open questions such as the exact function of CRISPR-T. At the same time, a switchable protease is a tool of great biotechnological interest. Therefore, the protease activity and the substrate specificity of CRISPR-Lon shall be characterized. We aim to investigate, whether CRISPR-Lon can be used in eukaryotic systems in order to negatively select such cells in which e.g., genome editing efforts did not deliver the desired result.

• DFG Programme: Research Grants