Functional Analysis Of CRISPR System And Application Of CRISPR-Cas9 In Myxococcus Xanthus

In the state of the harsh environment,prokaryotes are able to adapt and adjust to the changing world by developing new tactics to promote survival.One of the systems constituting a natural defense mechanism is the ability to distinguish self from non-self nucleic acids.Nucleic acids may be inserted into prokaryotic cells by infection,transduction,conjugation,or transformation,and may have harmful effects.The defense mechanisms include the restriction-modification,abortive infection,and surface exclusion systems,all of which act in an innate,non-specific way.Recently,it was reported that a unique defense system,involving Clustered Regularly Interspaced Palindromic Repeats-CRISPR-associated proteins?CRISPR-Cas?,provides the majority of bacteria and archaea with adaptive and hereditary immunity against this threat.This mechanism of immunity is based on short fragments of foreign DNA incorporated within the host's genome.After transcription,these fragments guide protein complexes targeting foreign nucleic acids and promoting their degradation.Till today,the CRISPR system has been supposed to play a role in chromosome rearrangement,DNA repair,gene expression regulation,spore and biofilm formation,pathogen resistance acquisition,and horizontal gene transfer.One of the new gene editing techniques based on the type II CRISPR-Cas system,which consists of gRNA and Cas9 proteins that recognize the DNA sequence of the target site,can perform double-stranded DNA cleavage.With advanced research,CRISPR-Cas9 system and its derivative technology have been widely used in genomic editing of different species.To date,the most well-developed methodsare based on the type II CRISPR-Cas effector complex,mainly because of its simplicity,that consists of Cas9 and sgRNAand can perform DNA cleavage.In addition,CRISPR-Cas9 system and its derivative technology?CRISPRi and CRISPRa?have been widely used in genome editing of different species.Myxobacteria are gram-negative bacteria belong to the delta group of proteobacteria that are noted for complicated multicellular social behavior and the excellent ability to produce abundant secondary metabolites.Myxobacteria harbor huge genome which have a large number of duplicated genes and horizontal gene transfer phenomenon,suggesting that the genome of myxobacteria easy to integrate foreign DNA and perform chromosome rearrangement,these phenomena are related to the function of CRISPR system.Through the analysis of sequenced myxobacterial genome by CRISPR database,we found two strains of sorangium?S.cellulosum So ce 56 and S.cellulosum So0157-2?and five strains of myxococcus containing CRISPR systems.Among them,the genome of M.fulvus 124B02 contains a Cas operon with 61 tandem spacers distributed in CRISPR loci;M.fulvus HW-1 contains two complete Cas operons with 130 spacers;M.stipitatus DSM 14675 harbors two complete Casoperons with 79 spacers;as the model strain of Myxobacteria,M.xanthus DK1622 contains three complete Cas operons with 159 spacers totally.This study aims to investigate the function of CRISPR system of M.xanthus and establishes an efficient and rapid knockout system in myxococcus using CRISPR-Cas9 genome editing tool,along with using CRISPRa to perform transcriptional activation of low expression secondary metabolic gene cluster in myxococcus.In addition,this study analyzed the function of double-copy DNA polymerase III a subunit related to the replication of M.xanthus and illustrated the role related to environment adaptation and evolution.In order to explore the role of CRISPR system of M.xanthus in cell survival,multicellular morphology,and evolution,the CRISPR system of M.xanthus DK1622 was analyzed by CRISPR database within this paper.We described the structure and made classification of CRISPR and analyzed the encoding products of genes which were matching with the spacers.We found that the function of these genes was related to cell survival,phage infection and environmental adaption.Furthermore,the differences between mutant strains and wild strain in the growth condition and the social behavior were analyzed by knocking out each CRISPR-Casregion in order to explore the function of the CRISPR-Cas system in the strain.The deletion of CRISPR2 weakened the development and sporulation ability of the strain,and the deletion of CRISPR3 reduced the production of extracellular polysaccharide?EPS?of DK122 and enhanced the efficiency of the natural transformation.Deletion of its own CRISPR system explored how CRISPR affected its physiological characteristic and cleared the immune background for the introducing CRISPR-Cas9 genome editing tools into myxobacteria.M.xanthus DK1622 has been studied as a host for heterologous expression and has a clear genetic background.Its genome is large?9.14Mb?and nearly 1.4Mb came from horizontally transferred genes?HGT?,which encodes secondary metabolites.At present,the genetic manipulation of myxococcus is mainly carried out by transduction and electroporation,for example,plasmid pSWU30 based on attB site integration and plasmid pMiniHimar-lacZ can complete random gene insertion;The self-replicable plasmid pZJY41,which was isolated fromM.fulvus 124B02,it can be transferred from E.coli to myxococcus.The knockout of myxococcus was performed by plasmid pBJ113 which has Kanamycin resistance gene cassete and galK for negative selection.This method requires two-step screening,the first step was resistance screening for once homologous recombination strains and the second step was usage of galK resistance D-galto obtain deletion strains.Because of low flux and long knockout cycle,some gene and antisense RNA cannot be knockout,and the knockout of some genes caused changes in chromosomal structure and may bring phenotypic illusion,which limited the development of the study of M.xanthus.In recent years,a large number of studies have found that CRISPR-Cas system has great potential in gene editing.Especially the Type ? CRISPR-Cas9 system derived from Streptococcus pyogenes,because it requires only one protein?Cas9?to form a protein nucleic acid complex with the crRNA and tracrRNA chimera,and scan the genome to find a PAM sequence that is complementary to spacer,then the cleavage of the target gene forms DNA double strand breaks?DSBs?.This method is widely used in eukaryotes,as well as a few prokaryotes,to complete the precise mutation of single gene or multi-gene simultaneous knockout.In order to improve the genetic manipulation efficiency of myxococcus,we established a genetic manipulation tool based on the CRISPR/Cas9 system to delete the genes in the genome.We introduced the codon-optimized cas9 gene into DK1622 and ACRISPR mutant strains,and confirmed that the Cas9 was expressed and correctly folded using fluorescent report gene.Two methods have been developed to repair targeted DSBs.The first,non-homologous end joining?NHEJ?,which exists in eukaryotes and didn't depend on homologous templates.The Ku and LigD proteins of P.aeruginosa PAO1 and M.fulvus 124B02 were used to perform the role of NHEJ.After the target gene was cut by Cas9,the two ends were repaired and completed the deletion of differently sized target fragments.The second protocol was adding the homologous recombinant DNA template?HR?,andusing the homologous recombination to repair DSBs,complete the precise deletion of genes.This technique achieved a one-step knockout of the large gene in the M.xanthus DK1622,greatly reduced the time of genome editing and became a new tool for efficient genetic manipulation in myxococcus.Another notable application of the CRISPR-Cas9 system is using dead Cas9,which binds to the target gene without cleavage,to activate the expression of the target gene,called CRISPRa.The advantage of CRISPRa compared withthe overexpression of genes is that it can be activated at the transcription initiation site?TSS?of the gene for some difficult cloning or large genes.This method is using dCas9 fusedwith activating protein to recruit RNA polymerase.The usage of dCas9-activating factor focus on mammalian cells,such as VP64,p65AD,which were fused to dCas9 and lead by sgRNA.The case of CRISPRa usage in bacteria is limited,mainly in E.coli,Streptomyces coelicolor and Streptococczus pneumoniae gene activation.At present,the use of CRISPR for microbial secondary metabolic gene cluster activation has not been reported.As an excellent host for the heterologous expression of secondary metabolites of myxobacteria,M.xanthus DK1622 has short generation time?4h?,growth cell is dispersed and it can provide the necessary substrate and enzyme.In previous study of our lab,Zhu et al.integrated the 56-kb gene cluster for the biosynthesis of antitumor polyketides epothilones into M.xanthus genome by transposition insertion.Here,we firstly constructed dCas9-activating domain system in Myxococcus xanthus ZE9,which had expressed heterologously epothilone gene cluster.Then we designed different sgRNA to lead dCas9-activation domain to target on different locations for the two TSSs in the promoter of epothilone gene cluster.In this paper,we used co-subunit and a-subunit of RNA polymerase,global regulatory factor?a54?and ECF sigma factor?CarQ?from Myxococcus xanthus DK1622.In addition,we detected the production of epothilone and the expression level of every module of the gene cluster to compare the activation effect of different locations which sgRNAs lead to and different activation proteins used in this study.In addition,we changed the promoter of dCas9-activation domain to copper-inducible promoter which was found in Myxococcus xanthus DK1622 and further improved the production of epothilone.It was the first time to construct CRISPR-Cas9 activation system in myxobacteria,as well as the first time to enablethe secondary metabolic gene cluster and attain high production of relevant product in prokaryotes.It also provided a powerful tool for discovery of microbial secondary metabolites.In the study of the replication and repair system of M.xanthus,we found that all of the sequenced myxobacteria had two dnaE genes.DnaE is an alpha subunit of the tripartite protein complex of DNA polymerase III that is responsible for the replication of bacterial genome.The dnaE gene is often duplicated in many bacteria,and the duplicated dnaE gene was reported dispensable for cell survivals and error-prone in DNA replication in a mystery.In this study,we found that all sequenced myxobacterial genomes possessed two dnaE genes.The duplicated dnaE genes were both highly conserved but evolved divergently,suggesting their importance in myxobacteria.Using M.xanthus DK1622 as a model,we confirmed that dnaEl?MXAN₅844?was essential for cell survival,while dnaE2?MXAN₃982?was dispensable and encoded an error-prone enzyme for replication.The deletion of dnaE2 had small effects on cellular growth and social motility,but significantly decreased the development and sporulation abilities,which could be recovered by the complementation of dnaE2.The expression of dnaEl was always much higher than that of dnaE2 in either the growth or developmental stage.However,overexpression of dnaE2 could not make dnaEl deletable,probably due to their protein structural and functional divergences.The dnaE2 overexpression not only improved the growth,development and sporulation abilities,but also raised the genome mutation rate of M.xanthus.We argued that the low-expressed error-prone DnaE2 played as a balancer for the genome mutation rates,ensuring low mutation rates for cell adaptation in new environments but avoiding cells damage from high rate of mutation.