The Preliminary Studies Of CRISPR System Editing Phosphoglycerate Kinase Gene Of Brucella

CRISPR,an adaptive immune system derived from bacteria,is employed to edit gene in plants,animals and bacteria.The novel system CRISPR is consisted of cas protein that can cut the DNA like nucleases,gRNA that direct cas9 to cut the target gene precisely.Even though CRISPR has already edited genome of more than 16 kinds of bacteria successfully,it is still not reported to edit intracellular bacteria such as Brucella,legionella and et al.Brucellosis is very severe in China,especially in the north of China.Moreover,the infection mechanism remains unknown until now.More importantly,the efficient vaccine and diagnose have not been discovered at present.Based on the background,we investigate the possibility of CRISPR system in editing gene of Brucella.If gene-editing system CRISPR can be exploited to edit gene of Brucella,this system would provide the valid tool for vaccines and infecting mechanism of Brucella.The following data was got in our experiments.1,The pBBR2-cas9-gRNA1/2/3 CRISPR cleaving system(A)that targets PGK gene were constructed via the pBBRMCS-2 vector successfully Meanwhile,in order to overcome lackness of Non-homologous End Joining of Brucella,pBBR2-cas9-gRNA1/2/3-H editing system(B)that targets PGK gene is constructed by inserting upstream 1-kb and downstream 1-kb homology arms into pBBR2-cas9-gRNA1/2/3.Then A or B plasmid were transformed into Brucella by electroporating.Our data showed that Brucella containing A is not able to grow in tryptic soy broth(TSB)plate.Although several colonies of Brucella containing B can grow in the TSB plate,the results from sequencing demonstrated that the PGK gene of theses Brucella is not cleaved by CRISPR system.This experiment implies that the constitutive CRISPR/cas9 system fails to edit Brucella genome due to not repair the cleaving sites effectively.2,Because of the failure to edit gene of Brucella by constitutive CRISPR/cas9 system,we predict that one of possible reasons is off-target of cas9.So So pBBR2-Fn/LbCpf1-crRNA1/2 cleaving system(C,D)were constructed by replacing cas9 with more loyal Fn/LbCpf1,or Fn/Lbcpf1-crRNA1/2-H(E,F)were constructed by inserting upstream 1-kb and downstream 1-kb homology arms into pBBR2-Fn/LbCpf1-crRNA1/2.Then C,D,E or F plasmid were transformed into Brucella by electroporating,respectively.Our data showed that all of Brucella containing relative plasmid is not able to grow in TSB plate compared with control group,which can grow normally in the TSB plate.This experiment proposes that off-target is not the reason of CRISPR's failure to edit gene of Brucella.3,Due to failure to edit Brucella genome by CRISPR/cas9?CRISPR/ FnCpf1 and CRISPR/ LbCpf1,we futher hypothesized that another reason of failure is unsufficient time for recombination caused by continuous expression of cas9 and cpf1.So,we constructed IPTG-Inducible CRISPR/cas9 system(pBBR2-lac-cas9-gRNA1/2/3(G),pBBR2-lac-cas9-gRNA1/2/3-H(H)including homology arms of PGK)and temperature-inducible CRISPR/cas9 system(pBBR2-cits-cas9-gRNA2(J),pBBR2-cits-cas9-gRNA2-H(K)including homology arms of PGK).Then G,H,J or K plasmid were transfected into Brucella by electroporating,respectively.Our data showed that all of Brucella containing relative plasmid is not able to grow in TSB plate after inducing compared with control group.Interestingly,if the above Brucella was not induced by IPTG or temperature,these bacteria did not grow,either.This experiment suggests that the inducible CRISPR systems cannot provide sufficient time for repairing the cutting sites via recombination due to the leaking expression of cas9,which result in failure of CRISPR gene editing.This experiment also imply that the cas9 expression is toxic to Brucella cell even in trace expressionIn conclusion,we investigated the possibility of CRISPR gene-editing system based on current development of CRISPR.Our data demonstrated that traditional all-in-one CRISPR system still is not able to be applied into Brucella genome editing.The extract mechanism by which CRISPR fails to edit Brucella genome deserve exploring further in the future.