Structural And Functional Studies Of Cas7-11 Effector Proteins In The CRISPR Syste

Purpose and significance:The CRISPR（clustered regularly interspaced short palindromic repeats）-Cas（CRISPR associated protein）system is a highly adaptive natural prokaryotic defense system that can resist invading viruses and plasmids.The CRISPR-Cas system has undergone more than 20 years from its discovery to the determination of its function.Its basic structure and mechanism of action have gradually been elucidated in recent years.Current studies have shown that CRISPR-Cas systems in nature can be divided into the first and second categories according to the composition of effector complexes:one kind of CRISPR-Cas system uses multiple Cas protein effect complexes to recognize and cleave target nucleic acids,including typeⅠ,ⅢandⅣ;the second type CRISPR-Cas system uses a single large Cas effector protein to play a role,including typeⅡ,Ⅴand VI.Among them,the system targeting RNA includes typeⅢsystem in the first class and type VI system in the second class.Although the DNA targeting system has developed into a powerful genetic modification tool,the CRISPR-Cas system targeting RNA has only emerged as a programmable tool in recent years.As a powerful genetic tool for biomedical research and gene therapy,class II VI effectors composed of Cas13 protein and crRNA have occupied a leading position.At present,the"off-target effect"of this subversive technology has been one of the key obstacles to its clinical application,so there is an urgent need to find Cas effector proteins with both high specificity and low cytotoxicity.In 2019,typeⅢ-E CRISPR-Cas system was discovered.Subsequently,two studies identified the characteristics of typeⅢ-E CRISPR-Cas systems,which were called Cas7-11 by（?）zcan et al,and g RAMP by van Beljouw et al,and proved their applicability as highly specific programmable integrated RNA targeting systems.The discovery of this system provides a new tool for sequence-specific RNA targeting,which can be used for gene knockdown and gene editing,and the miss effect is negligible and the cytotoxicity is low.At the same time,another unique feature of this system is that it can couple with another Caspase-related protease system called TPR-CHAT,activate and specifically cleave downstream effector proteins,and then induce prokaryotic cell apoptosis and participate in the host virus immune process.Cas7-11 can form a stable complex with crRNA and TPR-CHAT,which is called Craspase complex.In view of the broad prospect of this system in biomedical and therapeutic applications,the Craspase complex is expected to become a tool for gene therapy and biomedical research,but little is known about its regulatory mechanism.In order to explain the evolution of effector proteins from multi-component to single component in the first category of CRISPR system,and to provide a new RNA editing tool CRISPR-Cas7-11 with high specificity and low cytotoxicity,and Craspase complex to further promote the development of RNA editing technology and clinical application,it is very important to understand its regulatory mechanism.Methods and results:In order to study the structural basis of Craspase complex regulation,the experiment studied the type III-E CRISPR gene locus from Desulfonema ishimotonii and obtained the gene sequences of Cas7-11 and TPR-CHAT.In order to obtain the Cas7-11-crRNA complex,the plasmid containing Cas7-11 gene and crRNA gene were co-transferred,the Cas7-11-crRNA binary complex structure with the resolution of 3.4（?）,and the Cas7-11-crRNA binary complex structure with the lack of Cas11domain with the resolution of 2.5（?）by cryo-electron microscope.In order to obtain the Cas7-11-crRNA-TPR-CHAT complex,two vectors were constructed for co-expression and protein purification.Two kinds of structures were also obtained by cryo-electron microscopy,including the ternary complex structure of Cas7-11-crRNA-TPR-CHAT with the resolution of 2.8（?）,and the Cas7-11-crRNA-TPR-CHAT_NTD ternary complex structure lacking part of the TPR-CHAT domain with the resolution of 2.7（?）.New insight:First of all,concatenating multiple subunits into a single subunit can make expression and assembly more efficient,and has the potential to demonstrate the delivery advantages of single subunit systems（such as Cas9）.In addition,this system has been proven to have no off-target activity after targeting RNA,greatly reducing its toxicity in cells,and has great potential for future applications,along with the Cas13 protein,as a RNA editor.Finally,this system has a naturally coupled protease system,which may lead to the development of many new tools related to protein-directed degradation in the future,with great prospects.Moreover,the activity of Craspase in eukaryotic systems,especially in mammalian cells,can be used to help resist pathogen invasion or assist in cancer treatment.