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

CRISPR （Clustered Regularly Interspaced Short Palindromic Repeats）是一种在许多细菌和古细菌中发现的适应性免疫系统。它以病毒核酸的短片段（30-40个碱基对）的形式，将过去病毒攻击的“记忆”储存在宿主细菌的基因组中。这些“记忆”被转录并整合到rna -蛋白质传感器复合物中。在再次攻击的情况下，这些传感器复合物可以检测并摧毁相应的外来核酸。近年来，大量不同的CRISPR系统被发现和研究。事实证明，III型CRISPR系统特别复杂。在这里，传感器复合物在检测病毒核酸时合成所谓的环状寡核苷酸。这些信号分子激活效应蛋白，然后触发对病毒攻击的复杂反应。迄今为止，研究的主要是具有核酸酶活性的效应蛋白。它们以宿主自身的核酸为目标，从而使宿主细胞进入休眠状态。在这种状态下，细胞可以更好地抵御病毒的攻击。本申请的内容是对新发现的III型CRISPR效应物CRISPR- lon的研究。我们的初步工作表明，这个60 kDa的蛋白与较小的（34 kDa） CRISPR-T蛋白形成一个功能单元。与上述效应核酸酶不同，CRISPR Lon是一种蛋白酶，由具有4个AMP亚基的环状寡核苷酸激活。蛋白酶切割并可能激活CRISPR-T，这似乎是一种序列特异性核糖核酸酶。CRISPR- lon / t系统展示了III型CRISPR系统的全新方面，并且与真核天然免疫系统具有惊人的相似之处，其中蛋白酶如半胱天冬酶被用于快速响应“紧急”情况。本应用程序中的实验旨在充分表征CRISPR-Lon / -T系统，并回答诸如CRISPR-T的确切功能等开放式问题。与此同时，一种可转换的蛋白酶是一种具有巨大生物技术价值的工具。因此，需要对CRISPR-Lon的蛋白酶活性和底物特异性进行表征。我们的目标是研究CRISPR-Lon是否可以用于真核生物系统，以便负选择这些细胞，例如，基因组编辑工作没有提供期望的结果。

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.