A Complement III Gene Defective Pig Model Was Constructed Using CRISPR/Cas9 Technology

C3 plays a central role in activating of complement system through classical,alternative complement activation pathways,even mannose-blinding lectin(MBL)pathway.It has attracted much attention in recent years.Intensive studies have been conducted by using C3 knockout mice models which are helpful for elucidation of the function of the complement protein C3 in inflammatory response,immune defense,immune regulation.C3 gene mutation or deletion can also lead to immune deficiency,induced immune diseases and bacterial infections.Studies have shown that complement C3 gene deletion can prolong the survival rate of skin grafts,indicating the importance of the complement system for xenogeneic organ transplantation.With regard to anatomy,physiology and immunology,pigs are more similar than rodents to humans.The purpose of present study was to generate the first C3 gene knockout piglets using Clustered regularly interspaced short palindromic repeats/Cas9(CRISPR/Cas9),which will be ideal large animal models for further study the role of complement protein C3.In this study,the sgRNAs were designed based on the sequence of 26th exon of the Bama mini-pig C3 gene.The C3-Cas9 targeting plasmid was co-transfected with a neomycin-expression plasmid(pCMV-tdTomato)expressing the red fluorescence into porcine fetal fibroblasts(PFFs)by nucleofection.After G418 selection,the knockout cell colonies were screened by sequencing.The clonal pigs with complement C3 knockout were constructed by using somatic cell nuclear transfer(SCNT)technique.A total of 19 C3 knockout(KO)piglets were produced and their genotype were sequenced.46 cell colonies were analyzed by PCR sequencing.The results showed that 39 colonies bore mutagenesis in the C3 target site,indicating the targeting efficiency reached 84.7%(39/46).Moreover,45.7%(21/46)of colonies were biallelic knockout.Three or four biallelic modified colonies with different genotypes were selected as nuclear donors for somatic cell nuclear transfer into six recipient pigs.Pregnancy was established in two of six transfers.One pregnancy was maintained to term,giving birth to six piglets.Another embryo transplantation has been done in three recipients and two pregnancies were maintained to term and 13 live-born piglets were deliverd.Taken together,19 C3 gene knockout piglets were generated by the highly efficient CRISPR/Cas9 gene targeting system in this study.These C3 KO piglets could be utilized as a valuable large animal model for the elucidation of the roles of C3.