Stduy On Gene Editing Of Porcine MRC1 Mediated By CRISPR/Cas9 System To Reduce PCV2 Duplication

CRISPR/Cas system is an adaptive immune system widely existing in bacteria and archaea.Type II CRISPR/Cas system became one of the most effective genome editing tools today due to the advantages of simple operation and efficient editing.Porcine circovirus type2（PCV2）is a DNA virus with small genome,but it has high evolution rate.It causes post weaning multisystemic wasting syndrome and other porcine circovirus related diseases（PCVAD）,which caused great losses in pig industry.Thus,improving the resistance of pigs to PCV2 by CRISPR/Cas9 system is of great significance to the development of animal husbandry.Mannose receptor C1 gene（MRC1）is a homonymous member of the mannose receptor family.In the previous study,the Laiwu pigs,which is an excellent local breed in China,exhibited stronger PCV2 resistance capacity than the commercial one.Nucleotide polymorphism analysis showed a 14 bp fragment insertion at-864 upstream of the transcription initiation site of porcine MRC1 gene that significantly increased the promoter activity.The genotype frequency of 14 bp fragment insertion homozygote were evaluated and the data showed it was significantly higher in Dapulian and Laiwu pigs than in commercial one,which were related to the resistance to PCV2 infection of pigs.In this study,CRISPR/Cas9 system was used to insert the 14 bp nucleotide sequence into the upstream of MRC1 gene in PK15 cells to increase the expression of MRC1 gene and decrease the PCV2 duplication ability.And Cre-LoxP system was used to delete marker genes in CRISPR/Cas9 system mediated gene editing,in order to reduce the biosafety risks caused by foreign gene insertion.The main results are shown as follow:（1）Cre recombinase was inducibly expressed and purified.SDS-PAGE analysis showed the optimal condition of Cre recombinase was 1.0 m M IPTG at 37 ℃ for 9 h.After the Cre recombinase had been purified,the His labeled was used to assess the Cre recombinase,and the result of Western Blotting showed that the Cre recombinase reacted specifically with the His labeled antibody,and there was an obvious band at 38.5 k D.（2）The p LV-LoxP-m Cherry-puro-LoxP vector was constructed and verified.The p LV-LoxP-m Cherry-puro-LoxP vector was successfully constructed,which contains the multiple cloning sites,puromycin resistance gene and the same direction LoxP sequences.The constructed lentiviral vector was packaged and the virus were concentrated,then used for PK15 cells infection.PK15-m Cherry cells were obtained after screened by puromycin.The Cre-LoxP system was then assessed in the monoclonal cells by being treated with Cre recombinase and Cre plasmid,separately.Flow cytometry showed the Cre recombinase efficiency is obviously lower than the Cre plasmid,and the expression efficiency of the Cre plasmid was 16.2%.（3）The homologous recombinant vector and six targeting vectors of MRC1 gene were constructed.The homologous arms were designed and the MRC1 homologous recombination vector was successfully constructed by the differential sequence of MRC1 gene promoter.Six potential target vectors of the homologous arms were insert into the Lenti CRISPR v2 vectors.The targeting activity of the targeting vectors were identified by the T7E1 enzyme.Finally,sg RNA 3 and sg RNA 4 showed the highest activity of 48% and 49%,respectively.（4）The 14 bp upstream of the transcription initiation site of MRC1 gene was knocked into PK15 cells by CRISPR/Cas9 system.The homologous recombinant vector and two targeting vectors of MRC1 gene were respectively packaged with lentivirus to infect PK15 cells.Monoclonal cells were screened by puromycin.Finally,seventeen monoclonal cells were obtained,of which three were positive which was identified by PCR.And the Cre plasmid was used to delete marker genes.（5）The antiviral ability of MRC1 edited cells was identified.Compared with PK15 cells,the expression of MRC1 protein in MRC1 edited cells increased significantly（P < 0.05）and the viral load of PCV2 obviously decreased（P < 0.01）after infected with PCV2,which indicating the insertion of MRC1 gene promoter fragment could significantly decrease the PCV2 duplication ability.In conclusion,Cre recombinase was successfully expressed and purified in this study.The p LV-LoxP-m Cherry-puro-LoxP vector was constructed,and the Cre-LoxP system was assessed by being treated with Cre recombinase and Cre plasmid.The 14 bp upstream of the transcription initiation site of Laiwu pig’s MRC1 gene was knocked into PK15 cells,so that the expression of MRC1 protein increased and the PCV2 duplication capability decreased.This study lays a foundation for the anti-PCV2 gene editing pigs production.