Generation Of NRAMP1 Precise Knockin Cattle Via The CRISPR/Cas9 System

Tuberculosis(TB)is a globally devastating zoonotic disease caused by the infection of Mycobacterium tuberculosis.There are as many as 2 billion potential infection carriers around the world,among these about 5-10 percent would show clinical sumptoms and caused about 1.5 million cases of death each year.Bovine tuberculosis is caused by Mycobacterium bovis(M.bovis),it can also infected human beings through direct contact with the non-sterile meat and milk products.There was no effective strategies to control the M.bovis infection during the cattle rearing period in the less-developed areas of Africa,Latin America,and Asia due to the lack of efficient vaccines.Thus,M.bovis caused significant economic hardship for the global public health and agriculture.Researchers found that the host resistance to M.bovis infection is associated with a variety of genetic factors,among these the expression level of natural resistance-associated macrophage protein-1(NRAMP1)gene was demonstrated have impact on the host susceptibility to M.bovis.Our previous work showed that the host macrophages can effectively control the proliferation of M.bovis after the high expression of bovine NRAMP1 gene in RAW264.7 cell lines.Thus,this study aims to generate transgenic cattle with enhanced resistance to M.bovis through the insertion and over-expression of NRAMP1 gene.The CRISPR/Cas9 system is a widely utilized platform for transgenic animal production in various species,although its off-target effects should be addressed.Several applications of this tool have been proposed in model animals but remain insufficient for transgenic livestock production.Especially,coverages of functional exogenous gene insertion in mammalian genome are very limited.In this study,we report the first application of single Cas9 nickase(Cas9n)to induce gene insertion at a selected locus in cattle with genome-wide reduced off target effects.Through somatic cell nuclear transfer,we finally obtained 11 NRAMP1 transgenic cattle which can enhance the expression of NRAMP1 gene and the subsequent Activation of apoptosis signaling pathways and result in the increased resistance to tuberculosis.The main contents of this research are as follows:1.An intergenic region between the fascin actin-bundling protein 1 gene(FSCN1)and the actin beta gene(ACTB)on chromosome 25(F-A locus)was selected for gene target.To implement the CRISPR/Cas9 system for cattle genome editing,an evaluation model was developed for sgRNA selection based on the open-source website ZiFiT and an effective web-based off-target prediction tool termed Cas-OFFinder.Finally,14 potential target sites with less predicted off target sites were selected for the construction of Cas9 target plasmids and subsequent DNA cleavage efficiency detection in BFFs via Surveyor nuclease assays.Then,the gene-targeting donor vectors were constructed for the corresponding target sites to confirm the recombination efficiency induced by WT Cas9,single Cas9 n,and paired Cas9 ns.2.In the current study,chromatin immunoprecipitation followed by sequencing(Ch IP-seq)was performed on nuclease dead Cas9(dCas9)for protein binding site detection in BFFs for the first time.Thus the performance of dCas9:sgRNA binding to the chromatin structure and the main influence factors were indicated.Moreover,we clearly identified the main potential off-target sites and set up a criteria for the off-target effects assessment during gene target successfully.3.We constructed the Cas9 and Cas9 n target plasmids and the corresponding donor vectors pNRAMP1-eGFP-P2A-Puro-1/2.After co-transfection and 8–10 days of puromycin selection,the firmly transfected colonies were screened and analyzed by junction PCR,Sanger sequening,Southern blot and absolute quantitative PCR.Then the positive NRAMP1 transgenic colonies were used as donor cells for somatic cell nuclear transfer(SCNT),and finally obtained 11 NRAMP1 transgenic claves.Further test on the transgenic cows indicated that a single Cas9n-induced single-strand break can stimulate the insertion of NRAMP1 gene with genome-wide reduced off-target effects.4.To estimate the ability of NRAMP1 transgenic cows to resist M.bovis infection,transgenic macrophages were challenged with M.bovis in vitro.The results of Western blot,CFU test and flow cytometry analysis showed that the transgenic macrophages could limit the multiplication of M.bovis through the robust expression of NRAMP1 and the activation of characteristic apoptosis after infection.After in vivo challenge experiment on the experimental control normal and NRAMP1 transgenic cows,the IFN-? release assays(IGRAs)and MTB-specific enzyme-linked immunospot(ELISPOT)assay further demonstrated that the transgenic cattle exhibited increased resistance to M.bovis.In summary,we demonstrated that a single Cas9 n can be used for gene insertion at a selected target site in the cattle genome and that this method is advantageous in terms of avoiding additional indel mutations.The resulting transgenic cattle exhibited increased resistance to Mycobacterium bovis infection.Our study provides an avenue to develop the CRISPR/Cas9 system for agriculture applications.