Targeted Editing Buffalo BMP15 And GDF9 Via CRISPR/Cas9 System

Buffalo is an economically important livestock species in south China, which dued to provide a high quality of milk, raise farms and find them hardy. And so, according to FAO’s latest estimates, the buffalo is one of the most potential development species. However, the low fertility of buffalo, characterized by low conception rate, seasonality, anestrous and delayed puberty, is in urgent need of improvement through the propagation of superior germ plasm by the modern reproductive technologies. BMP 15 and GDF9 play an important role in follicular development. BMP15 and GDF9 heterozygous sheep which lambing rate are significantly higher than the wild type. And Clinical medicine data aslo suggest that BMP 15 and GDF9 mutations women also significantly affect pregnancy. Accordingly, BMP 15 and GDF9 become our candidate genes to improve fertility buffalo. In order to study the feasibility of improving buffalo reproductive performance through Crispr/Cas9 system. The main results of this study are summarized as follows:1. Cloning and analysis the buffalo BMP15 and GDF9 genes, and expression pattern of folliculogenesis genes in follicular developmentPrimers were designed to amplify buffalo BMP 15 and GDF9 CDS and BMP 15 complete gene sequences. The results revealed that the CDS length of BMP 15 and G DF9 were 1185bp and 1364bp, respectively, and complete gene sequences length of B MP15 was 6490bp. Cloning genes were homologous to bovine,99% and 98%, respect ively. Meanwhile, the proteins informations were also analysised using online Bioinfor matics, the results revealed that there was a conservative TGF-β domian in 290AA-36 8AA (BMP15) and 389AA-449AA(GDF9). BMP6, BMP4, Kit, C-kit and BMP15 were expressed all follicular development through qPCR. In this study, I saw that Kit was st rongly preferred at fetus ovary, In contrast to BMP15. Additionally, we saw a preferen ce for BMP4 and BMP6 in antral follicle.2. Optimization CRISPR/Cas9 system about gene targeted editingBased on buffalo BMP 15 and GDF9 gene from cloning, I designed and constructed six gRNAs and corresponding reporter, respectively. The expression of EGFP in the reporter was significantly increased than control group when combination b-BMP15-gRNA2/4/5 or b-GDF9-gRNA4/5 with Cas9 by fluorescence microscope and flow cytometry. Analysis of fluorescence intensity at different times, Gene targeted editing is time dependent, and the expression of EGFP was highest after transfection 48h. Of note, a ratio of Cas9:gRNA at 1:4-8 have an optimal condition to induce gene targeted mutation.3. Targeted editing human genome by Crispr/Cas9 systemBased on human BMP 15, GDF9, CN2 and involved DNA repairnon-coding gene miRNA182 and miRNA155 from NCBI, I designed and constructed six h-BMP15 gRNAs, two h-GDF9-gRNAs, three h-mi 182-gRNAs, two h-mi155-gRNAs and two h-CN2-gRNAs, respectively. Next, when examining nucleotide preferences for active sgRNAs at every position across the different sgRNA sequence, Specifically, I looked for statistical enrichment or depletion of sgRNAs with a given sequence feature among the 20 active sgRNAs for the different gene target. Within the sgRNA sequence, the most significant differences appeared at position 20, the nucleotide immediately adjacent to the PAM. I saw that guanine and cytosine were strongly preferred at position20. Additionally, we saw a preference for adenylate from position 9 to 15 in the sgRNA sequence. In addition, I analyzed the efficiency of miRNA182-gRNA and miRNA155-gRNA2 were 16.6% and 20%, respectively. Interestingly, the efficiency of miRNA155-rRNA3 was 100%. It suggested us different genetic 1-oci in the genome had significantly different editing efficiency.4. Targeted editing buffalo genome by Crispr/Cas9 systemBuffalo BMP 15 and GDF9 were precise edited by Crispr/Cas9 system. To selecte b-BMP15-gRNA4/5 and b-GDF9-gRNA4/5 from each gene, respectively, I analyzed the gene mutation efficiency of the CRISPR-Cas systems. The efficiency of b-BMP15-gRNA4/5 were 44.4 and 55.6%, respectively. And the efficiency of b-GDF9-gRNA4/5 were 30.8% and 40%, respectively. Next, to analyze the double-gene mutation efficiency of the b-BMP15-gRNA4/b-GDF9-gRNA5 or b-BMP15-gRNA5/b-G DF9-gRNA4 were 37.5%(BMP15),10%()GDF9 and 30%(BMP15),40%(GDF9), respectively. In additional, efficient generation of large-scale (5.4Kb) buffalo BMP 15 gene deletion using two candidate gRNA. To detect off-target effects of the Crispr/Cas9 system in BFF cells, I screened the BMP 15 SNP site which sited PAM motifs. gRNA mediated Cas9 editing efficiency significantly was reduced when PAM altered.5. To explore the DNA repair pathway choiceThe insertion of precise genetic modifications through CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the hig-her efficiency of the nonhomologous end-joining (NHEJ) pathway.However, cell cycle and chromosome epigenomics information significantly contral DNA repair pathways choice. The study found that TSA significantly reduced the expression of EGFP in the reporter, but 5’-AZA and MG132 had no effect. Meanwhile, TSA was combined with 5’-AZA or MG132 had not further reduced the expression of EGFP fluorescence in the reporter. The expression of BRCA1 that inhibits NHEJ pathway was significantly i-ncreased when treated BFF, Bcap37 and 293T cells with 500nm/ml TSA by qPCR an dWestern bloting.Conclusion:This project demonstrated that buffalo genome was hihg-efficiency and precise editing via CRJSPR/Cas9. In additional, the double genes targeted mutation were gotten in the buffalo BFF through CRISPR/Cas9. The experimental, therefore, could be utilized in future experiments aiming to improve buffalo reproductive ability.