Structural Basis Of CRISPR-Cas12j2 Effector Protein

The CRISPR-Cas system is an RNA-guided adaptive immune system in prokaryotes that protects the host from invasion by foreign nucleic acids.The CRISPR-Cas system consists of clustered regularly interspaced short palindromic repeats（CRISPR）and CRISPR-associated proteins（Cas）.CRISPR-Cas systems can be divided into two classes（class1 and class2）,the class1 of CRISPR-Cas systems utilize crRNA and multiple small Cas effector proteins to form interfering modules,including types I,types III and types IV,while class 2 CRISPR-Cas systems consist of crRNA and a single large protein,including types II,V,and VI,such as the CRISPR-Cas9 system and the type V CRISPR-Cas12a system,showing programmable nuclease activity,have been successfully used in the field of gene editing.CRISPR-Cas12j is a family of small RNA-guided endonucleases recently discovered from bacteriophage.These Cas proteins contain 700-800 residues,provide a small-scale tool for gene editing tools.Cas12j2 is an RNA-dependent DNase that belongs to the second largest class of type V CRISPR-Cas systems.In order to study the molecular mechanism of how Cas12j processes pre-crRNA and cleaves target DNA,we analyzed the catalytic state of the Cas12j2-crRNA-DNA ternary complex with high-resolution crystal structure and performed biochemical experiments.1.The structure of the ternary complex shows that the Cas12j2 effector protein consists of the REC lobe and the NUC lobe,the REC lobe consists of the REC1,REC2 and OBD domains,and the NUC lobe consists of the Ruv C domain and the ZR domain.The spacer region of crRNA and the prospacer of TS form a 16 bp heteroduplex,which together with NTS passes through a central channel surrounded by REC1,REC2 and Ruv C domains.2.The active site of CRISPR-Cas12j processing pre-crRNA is the same as the active site of cleaving target DNA.In this study,the structure of the Cas12j2 effector protein was analyzed and combined with in vitro biochemical experiments,it was identified that the key amino acids in the enzymatic activity center of processing pre-crRNA and cleaving target DNA are D394,E606,D695 located in the Ruv C domain3.The PAM sequence and seed sequence play an important role in the recognition of target DNA or target RNA by other CRISPR-Cas systems.This study demonstrates that both the structure and sequence of the PAM region and correct base pairing within the seed region（A（1）-T（8））are indispensable for Cas12j effector cleavage of target DNA.4.The crRNA is transcribed into pre-crRNA by the CRISPR sequence and processed into mature crRNA by the Cas12j2 effector protein.This study proves that nucleotides A（-2）and G（-17）-A（-20）in the repeat region are crucial for the processing and maturation of pre-crRNA,and the amino acid residue R139 of REC1 plays an important role in the recognition and cleavage of target DNA by Cas12j2 protein.5.Pre-crRNA processing experiments and target DNA cleavage experiments using different metal ions proved that the activity of Cas12j2 protein on pre-crRNA processing and target DNA cleavage depends on divalent metal ions(Mg²⁺,Mn²⁺).Our research resolves the high-resolution crystal structure of the Cas12j2-crRNA-ds DNA ternary complex,combined with in vitro biochemical experiments,the enzymatic active centers of pre-crRNA processing and target DNA cleavage are revealed,which can promote the understanding of the working mechanism of the CRISPR-Cas12j system and provide a more sufficient theoretical basis for the application of the CRISPR-Cas12j system in gene editing.