Structural Insights Into CRISPR-Cas12i1 Effector Guided DNA Cleavage

Being under the constant pressure of arms race with the exogenous mobile genetic elements（MGE）such as bacteriophages,prokaryotes have evolved a number of mechanisms to fight against invasions.One such defense mechanism is a form of acquired immune system known as CRISPR-Cas,which consists of a highly structured CRISPR array（clustered regularly interspaced short palindromic repeats）and genes encoding Cas（CRISPR-associated）effector proteins distributed near the array.It can perform multiple gene editing and modification functions.Based on properties of Cas effectors involved in cr RNA（CRISPR RNA）processing and nucleic acid interference,currently known CRISPR-Cas systems can be grouped into class 1 and class 2 systems.Class 2 CRISPR-Cas systems complete the interference progress using single Cas effector proteins,such as Cas9 and Cas12a（originating from type II and type V-A systems,respectively）that cleave double-stranded DNA,and Cas13 system of type VI systems that exclusively cleave single-stranded RNA.Cas12i is the effector protein of the newly reported type V-I CRISPR-Cas systems which processes pre-cr RNA autonomously and specifically cleaves ds DNA substrates.Cas12 i is also characterized by a relatively small size,programmable regulation and markedly different efficiencies in cleavage of two strands of target DNA,which altogether makes it a promising new candidate for application in high-fidelity genome editing.This study reports and analyzes the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage,followed by systematic biochemical experiments to elucidate the mechanisms underlying the target DNA duplex unwinding,R-loop formation and cleavage.Cas12i1 protein has a bilobed architecture,in which the REC lobe included the Helical-I domain and PI domain（PAM interacting domain）,and the NUC lobe included the WED domain（wedge domain）,Ruv C domain（nuclease domain,including Ruv C I-III）,Helical-II domain and Nuc（alkaline Nuclease）domain.The T-shaped R-loop structure composed of mature cr RNA and target ds DNA is sandwiched inside the positively charged channel formed by the two lobes.A key structural motif termed the zipper motif（residues 160–177 of the Helical-I domain）was identified to facilitate target ds DNA unwinding.In vitro biochemical experiments indicated that three catalytic residues D647,E894 and D1074 in the Ruv C domain are essential for the NTD strand cutting,and amino acid residues W915,H946,N649,R652,R962 had different effects on the cleavage activity.Moreover,a loop region（residues724–737）in the Helical-II domain facilitates the loading of TD strand beyond the cr RNA-target DNA strand heteroduplex into the Cas12i1 DNase active site.In addition,biochemical experiments indicated that substrate cleavage by Cas12i1 is temperature and metal-ion dependent.Finally,based on the results of this structural and biochemical study,a multi-step model is proposed to explain the process of catalytic activation of Cas12i1.This study will provide valuable information for rational engineering of the CRISPR-Cas12 i systems with the purpose of improving their fidelity and efficiency in future applications.