Structural Insights Into Cas13b-guided CRISPR RNA Maturation And Recognition

CRISPR-Cas systems are a form of RNA-guided adaptive immunity,protecting bacteria and archaea against invasive mobile genetic elements.They allow manipulation of multiple genomic sequences to achieve genetic alterations or interventions.CRISPR-Cas systems exhibit great diversity,which lead to a classification system based on the presence of distinct signature Cas proteins.Class 1 systems(including type I,III,and IV)utilize extensive Cas proteins and crRNA to form multi-subunit effector complexes.In contrast,class 2 systems(including type II,V,and VI)involve large single effector proteins in complex with crRNA for interference.The type II and V effectors,such as Cas9,Cas12a(Cpf1)and Cas12b(C2c1),have been engineered into powerful tools for genome editing.Cas13b,a newly identified class II type VI-B CRISPR-Cas effector,is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner.Here,to investigate how Cas13 b arranges its domains to process pre-crRNA,recognizes crRNA and settles the guide nucleotides for target recognition,we solved the crystal structure of Bergeyella zoohelcum Cas13b(BzCas13b)in complex with its crRNA.The architecture of BzCas13 b assumes a triangular domain distribution around the central L-shaped crRNA.The crRNA is bound within a positively charged channel formed by multiple domains of BzCas13 b.The repeat region of crRNA adopts a distorted A-form RNA duplex and is anchored inside the pocket formed by Helical-2,two RRI domains and the linker region.The spacer region of crRNA is wrapped by Helical-1,HEPN-1,RRI-1 and RRI-2 domains on one side,and by Helical-2 domain on the other side.BzCas13 b cleaves pre-crRNA at the phosphodiester bond connecting the two nucleotides located directly 3?-downstream of the repeat region.Residues K452 and R459 play an essential role for the pre-crRNA processing.Two RNase catalytic sites,one for pre-crRNA processing and one for target RNA cleavage,are independently located on different domains.The R116 A or R1177 A mutation was sufficient to abolish the target RNA cleavage activity.Moreover,in the binary complex,the catalytic residues from two HEPN domains remain too distant to cleave target RNA.Our findings reveal pre-crRNAprocessing site and crRNA recognition pattern of BzCas13 b,and builds a framework for developing the type VI-B CRISPR-Cas system into a tool for wide range of applications.