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

CRISPR(簇状规则间隔短回文重复序列)是一种适应性免疫系统，存在于许多细菌和古菌中。它将过去病毒攻击的“记忆”存储在宿主细菌的基因组中，以病毒核酸(30-40个碱基对)的短片段的形式存在。这些“记忆”被转录并合并到RNA-蛋白质传感器复合体中。一旦发生新的攻击，这些传感器复合体可以检测并破坏相应的外来核酸。近年来，人们发现和研究了大量不同的CRISPR系统。事实证明，第三类CRISPR系统特别复杂。在这里，当传感器复合体检测病毒核酸时，它会合成所谓的环状寡核苷酸。这些信号分子激活效应器蛋白，然后触发对病毒攻击的复杂反应。到目前为止，主要研究了具有核酸酶活性的效应蛋白。它们以宿主自身的核酸为靶标，从而使宿主细胞进入休眠状态。在这种状态下，细胞能够更好地抵御病毒的攻击。本申请的内容是对一种新发现的III型CRISPR效应器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蛋白形成了一个功能单位。与上述效应核酸酶不同的是，CRISPR Lon是一种由含有4个AMP亚基的环核苷酸激活的蛋白酶。这种蛋白酶切割并可能激活CRISPR-T，这似乎是一种序列特异性的核糖核酸酶。CRISPR-Lon/-T系统显示了III型CRISPR系统的一个全新方面，并与真核生物的天然免疫有着惊人的相似之处，在真核生物中，半胱氨酸天冬氨酸酶等蛋白酶被用来对“紧急”情况做出快速反应。本申请中的实验旨在全面描述CRISPR-Lon/-T系统的特征，并回答诸如CRISPR-T的确切功能等公开问题。与此同时，一种可切换的蛋白酶是一种极具生物技术意义的工具。因此，需要对CRISPR-LON的蛋白酶活性和底物专一性进行表征。我们的目标是调查CRISPR-Lon是否可以用于真核系统，以便负面选择这样的细胞，例如，在这些细胞中，基因组编辑工作没有产生预期的结果。

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.