Establishment Of An Endogenous CRISPR-based Genome Editing Method And Studies On Nucleic Acid Interference Of Type ?-B CRISPR System

CRISPR-Cas(clustered regularly interspaced short palindromic repeats-CRISPR-associated)systems are widespread in archaea and bacteria,which provide adaptive defence against invading genetic elements,including viruses and plasmids.In the latest classification scheme,CRISPR-Cas systems are classified into two classes with six types among which three main types(Type I,II and ?)are widely investigated.The Type II CRISPR/Cas9 systems are currently most used for genome engineering in many different bacteria and eukaryotes.Furthermore,Type I and Type II CRISPR-Cas systems have been explored for selectively removal of specific bacterial species as an antimicrobials.However,method hase not been reported for harnessing any Type I or Type ? CRISPR-Cas system for genome editing.Here,a method for genome editing using the endogenous Type I and Type ? CRISPR-Cas systems was developed in Sulfolobus islandicus.A plasmid carrying an artificial mini-CRISPR array would provide mature crRNAs(CRISPR RNAs)which can guide gene targeting or silencing.With a donor DNA for HR(Homologous Recombination)containing a non-target sequence added to the targeting plasmid,a novel type of genome-editing plasmid(pGE)was constructed.Transformation of a pGE plasmid would yield two alternative fates to transformed cells: wild-type cells are to be targeted for chromosomal DNA degradation,leading to cell death,whereas those carrying the mutant gene after HR would survive the cell killing and selectively retained as transformants.Using this strategy,different types of mutation were generated,including deletion,insertion and site-directed mutagenesis.We envision this method is readily applicable to different bacteria and archaea that carry an active CRISPR-Cas system of DNA interference.Studying molecular mechanisms of different CRISPR-Cas systems is the foundation of their application and has become one of the hottest research fields in contemporary biology.The type ? CRISPR systems are reported to be both RNases and RNA-activated DNases,which are significantly different from other CRISPR systems.Our previous research has revealed that the Sulfolobus islandicus type ?-B Cmr-? system has both RNA interference and transcription-dependent DNA targeting in vivo.To gain further insight into the involved mechanism,we purified the native Cmr-? effector complex from S.islandicus,carring crRNAs derived from the 43 nt SS1 spacer(target to the protospacer SS1 in lacS gene)and characterized it in vitro.We found that Cmr-? complex cleaved RNA complementary to crRNA present in the complex,and it also had the target RNA-activated ssDNA cleavage activity,the ssDNA destruction required mismatches between the 5'-tag of crRNA and the 3'-flanking region of target RNA.Moreover,the activated Cmr-? complex can destroy a large excess ss DNA substrate,which means Cmr-? system could rapidly degrede replicating viral DNA.To investigate the function of Cmr2? in the Cmr-? DNA interference,we constructed four mutants of HD domain and Palm domain of Cmr2?: denoted HD-M1(H14N,D15N),HD-M2(H14N,D15 N,K19A and I23A),Palm-M1(G666K,D667K),Palm-M2(662-IYlGGDDi LA-671 to AAlAAAAiAS).Our invader plasmid assay showed that the DNA interference in vivo was inactivated in HD-M2,Palm-M1 and Palm-M2 which is multiple mutation.However,double mutation of the HD motif(H14N and D15N)only abolished the DNase activity in vitro.These results indicated that both HD and Palm domains of Cmr2? are essential for the in vivo Cmr-? DNA interference,and the ssDNA cleavage activity we observed in vitro may be just mediated by HD domain.Otherwise,many detail features toward crRNA maturation and the mechanism of target capture in Type ? CRISPR-Cas system remain unknown.Here,mini-CRISPRs of SS1 spacer carring five different lengths(14,20,28,40 and 76 nt)were selected for producing corresponding native Cmr-? complexes.We found that two constant lengths of spacer(40 nt and 46 nt)generated by Type ?-B Cmr-? system independent on length of spacer,suggesting that the downstream repeat element was supplement into mature crRNA without processing.A construct containing only upstream repeat element and 40 nt spacer also produced mature crRNAs as same as WT.These data indicated that Cmr-? complex formation is launched from 5'-tag primary processed by Cas6 to produce two species of complexes and the 3'-end of crRNAs cleaved by an unknown nuclease(s).Furthermore,in the cleavage and binding assay of each Cmr-? complex,as the number of mismatches between 3'-end region of crRNAs and the 5'-end of target RNA increased,the amounts of free RNA substrated also increased.In the structural models of ?-B effector complexes,the 3'-end region of crRNAs is located in the Cmr1 subunit.Here,we constructed a deletion and two alanine-scanning mutants of Cmr1?: denoted ???1?,??1?-M1(W58A,F59A),??1?-M2(I52A,G54 A,R57A and R61A).The mini-CRISPR-based reporter gene assay showed that RNA interference was impaired in ??1?-M1 and almost eliminated in ???1? and ??1?-M2.While the subunits of the Cmr-? effector complex obtained from ??1?-M1 were very similar to the wild-type Cmr-? effector complex,the subunits of the Cmr-? effector complex purified from ??1?-M2 mutant resemble that of ???1?,in which Cmr1? protein is absent.RNA cleavage assay of these mutated Cmr-? effector complexes showed the consistent results with the observation that in vivo RNA interference.The target RNA-activated DNA cleavage assay indicated that both complexes from ???1? and ??1?-M2 were almost inactive in DNA cleavage,while ??1?-M1 had a very faintest ssDNA cleavage comparing with wild-type complex.Furthermore,electrophoretic mobility shift assay showed that both complexes from ???1? and ??1?-M1 were strongly impaired for the formation of a stable ternary complex with the target RNA.Taken together,Cmr1? functions in the formation of the target RNA-Cmr-? ternary complex,the two conserved hydrophobic amino acids are essential for the target RNA capture and four invariant amino acids may take charge of the strong interaction with crRNA for forming an intact Cmr-? effector complex including Cmr1?.