Preliminary Functional Study On Riemerella Anatipestifer CRISPR/cas-mediated Defense Against The Exogenous DNA

Clustered regularly interspaced short palindromic repeats?CRISPR?and CRISPR-associated?Cas?proteins provide acquired genetic immunity against the entry of exogenous DNA.The immune defense provided by various subtypes of CRISPR-Cas system has been confirmed and found to be closely associated with the formation of immunological memory in CRISPR arrays,called CRISPR adaptation or spacer acquisition.Subtype?-C CRISPR-Cas system is the most common subtype among type?systems;however,this system is the least well studied.A previous study revealed that Riemerella anatipestifer CH-2?RA-CH-2?encodes a type II-C CRISPR-Cas system.In this paper,the characteristics of the subtype system were investigated,including spacer acquisition?the adaptation phase?and the resistance against exogenous DNA?the interference phase?.The main results are as follows:1 Bioinformatics analysis of RA-CH-2 CRISPR-Cas systemCRISPR-Cas analysis of RA-CH-2 genome?NC₀20125.1?revealed the CRISPR array,gene sequences of cas9,cas1 and cas2,and the presumed sequences of leader and tracRNA.The architectural structure of RA-CH-2 CRISPR-Cas system was5'-tracRNA-cas9-cas1-cas2-leader-repeat1-spacer1···spacer15-repeat16-3',indicating that RA CRISPR-Cas system belonged to the II-C subtype system.2 Research on the essential gene for RA CRISPR-Cas adaptationMutant strains of RA-CH-2?cas1,?cas2 and?cas1-cas2 were constructed and introduced a shuttle plasmid or the plasmid expressing Cas1 or?and?Cas2.The results showed that cas1 or cas2 gene deletion of RA-CH-2 abolished spacer acquisition.This phenotype was restored only by the shuttle plasmid expressing both Cas1 and Cas2 in?cas1 or?cas2.However,when the site-directed mutation E149A,H206A,or E221A was introduced into Cas1 from the shuttle plasmid,spacer acquisition of?cas1 was abolished.Therefore,cas1 and cas2 were required for RA CRISPR-Cas adaptation,and the residues E149,H206 and E221 of Cas1 were identified as the functional sites for RA spacer acquisition.3 Research on the interaction of RA Cas1 and Cas2 in vitroThe recombinant plasmids for prokaryotic expression of RA-CH-2 Cas1,Cas2and Cas1-Cas2 were constructed,as well as the corresponding expression strains.And the rabbit polyclonal antibody against Cas1 recombinant protein was prepared.The expression of Cas1 or Cas1 mutant protein,and Cas2 protein was induced in BL21pET30a::cas1-cas2 by IPTG.The supernatants from the expression product were subjected to Ni-NTA pull-down assay and co-immunoprecipitation assay to analyze the interaction of Cas1 and Cas2 in vitro.The results showed that RA Cas1 interacted directly with Cas2 and formed a stable complex in vitro.The E149A,H206A or E221A mutation in Cas1 did not affect its interaction with Cas2.4 Research on dsDNA nuclease activity of RA Cas1DNase activity of the purified recombinant Cas1 protein or Cas1 mutant proteins was explored by adding these proteins to dsDNA substrates in the presence of KCl and metal ions(Mg²⁺,Mn²⁺,Ca²⁺,Fe³⁺,Zn²⁺,Co²⁺,Ni²⁺,Cu²⁺,or Ba²⁺)or EDTA.The results revealed that RA Cas1 was able to degrade circular and linear dsDNA,and Mg²⁺,Mn²⁺,Co²⁺or Ni²⁺assisted RA Cas1 in the cleavage of dsDNA.Metal-ion preference of RA Cas1 nuclease is dependent of salt concentration.Metal-ion chelation by EDTA inhibited Cas1-mediated degradation of dsDNA substrates.Alanine substitution of the residue E149,H206 or E221 of Cas1 inhibited the DNase activity.These data indicated that RA Cas1 was a metal-dependent(Mg²⁺,Mn²⁺,Co²⁺or Ni²⁺)nuclease,and the residues E149,H206 and E221 were the active sites for RA Cas1 nuclease activity.5 Research on the role of RA Cas9 in the CRISPR-Cas adaptationThe role of RA Cas9 in the CRISPR-Cas adaptation phase has been assessed by gene deletion and site-directed mutagenesis.?1?RA-CH-2?cas9 mutant strain was constructed and introduced a shuttle plasmid or the plasmid expressing Cas1,Cas2 or?and?Cas9.The results showed that cas9 gene deletion of RA-CH-2 abolished spacer acquisition,thus cas9 was required for the adaptation of RA CRISPR-Cas system.This phenotype was restored only by the shuttle plasmid expressing Cas1,Cas2 and Cas9 in?cas9.?2?The predicted active site of Cas9?D8 or H792?was mutated in the shuttle plasmid to alanine,and the ability of acquiring new spacers was enhanced in?cas9 carrying this plasmid,suggesting that the predicted nuclease activity of RA Cas9 was not required for the adaptation.6 Research on the inference RA CRISPR-Cas systemPlasmid loss assays and phage infection assays were performed to assess the defense against exogenous DNA provided by RA CRISPR-Cas system.?1?The plasmid loss rates of?cas1,?cas2 and?cas1-cas2 were significantly lower than that of the wild-type strain,suggesting that the active adaptation contributed to RA CRISPR/Cas-mediated interference with the exogenous plasmid.?2?Deletion of RA cas9 gene significantly decreased the plasmid loss rate,suggesting that RA Cas9 was involved in CRISPR/Cas-mediated interference with the exogenous plasmid.?3?The mutations D8A and H792A in RA Cas9 were introduced into the plasmid pLMF03::cas1-cas2-cas9,and the plasmid loss rate of?cas9 carrying the plasmid was significantly decreased,suggesting that the residues D8 and H792 of RA Cas9were the functional sites for the interference with the exogenous plasmid.?4?After 8 h in a mixed culture containing virulent bacteriophages,?cas9 carrying the plasmid pLMF03::cas1-cas2-cas9 began to recover their growth,and changed from declining to logarithmic growth.However,when residues D8 and H792 of Cas9 in the plasmid were mutated to alanine,the growth of the strain entered the decline phase after being mixed with the phage for 4 h.These results revealed that Cas9 was the key component for RA CRISPR/Cas-mediated bacteriophage resistance,and the residues D8 and H792 of RA Cas9 were the functional sites for the resistance.?5?A bacteriophage-insensitive mutant strain of RA-CH-2 was isolated,which incorporated a new spacer that matched the phage DNA in CRISPR1 array and escaped RA-CH-2phage infection,suggesting that the bacteriophage resistance was provided by RA CRISPR-Cas system.In summary,cas1,cas2 and cas9 are required for spacer acquisition of RA CRISPR-Cas system.RA Cas1 is able to degrade DNA and the nuclease activity of RA Cas1 is essential for spacer acquisition.The residues of D8 and H792 of RA Cas9are essential for the resistance to plasmid and bacteriophage,but dispensable for the adaptation.The CRISPR-Cas system allowed RA to escape phage infection by integrating new spacers that match the phage DNA.This study provides the first insight into the structure and function of RA CRISPR-Cassystem and the mechanism of the system-mediated immune defense against the exogenous DNA.