Structural Insights Into CRISPR-cas13d Effector

CRISPR-Cas systems are a form of RNA-guided adaptive immunity that protects host bacteria and archaea from phages and other foreign nucleic acids.Identified CRISPR-Cas systems display considerable diversity and can be divided into two classes.Class 2 CRISPR-Cas systems use a single multi-domain Cas effector in complex with cr RNA to accomplish RNA-guided cleavage of target nucleic acid sequences.Currently,modified versions of the class 2 type II CRISPR-Cas9 system and type V CRISPR-Cas12 a system are being widely used as gene editing tools.Cas13d is a family member of the class 2 type VI CRISPR-Cas systems.Because of its small size,absence of PFS(protospacer flanking sequence)constraints,and efficient target RNA cleavage activity,Cas13d has great application potential in the field of RNA editing.In this study,the high-resolution crystal structure of the uncultured Ruminococcussp.Cas13d(UrCas13d)in complex with its cognate cr RNA was analyzed,followed by systematic functional,mutational and biochemical experiments.The mature cr RNA was found to bind to positively charged central channel formed by the compact bilobed structure of UrCas13d.Two hydrated magnesium ions help to stabilize the conformation of the cr RNA repeat region.Biochemical experiments indicated that divalent metal ions are not required for pre-cr RNA(precursor CRISPR RNA)processing of UrCas13d,but are critical for target RNA cleavage.The sequence and conformation of nucleotides U(-8)～C(-1)of the cr RNA repeat region,which interact with one of the hydrated magnesium ions,are indispensable for target RNA cleavage and are specifically recognized by UrCas13d.Moreover,the active site for pre-cr RNA processing was pinpointed to the HEPN-2 domain(higher eukaryotes and prokaryotes nucleotide-binding domain)of UrCas13d,and amino acid residues R802 and K905 from the active site were shown to be essential for pre-cr RNA processing.Single-point mutations of the key amino acid residues mediating target RNA cleavage(R288A or H828 A from the two HEPN domains)resulted in complete loss of the target RNA cleavage activity.Finally,mismatch tolerance between cr RNA spacer region and target RNA sequences was investigated,finding that correct base pairing within two sub regions of the spacer region was essential for target RNA cleavage.In this study,the structure and function of UrCas13d-cr RNA binary complex are analyzed in detail,which provides a theoretical basis for rational engineering and future application of CRISPR-Cas13d systems.