The Effects Of Wnt16 Gene Knockout Using CRISPR/CAS9 On Skeletal Development In Zebrafish

Osteoporosis is a disease characterized by low bone mass and microarchitectural deterioration of bone tissue leading to increased risk of fracture. Osteoporosis was defined clinically through the measurement of bone mineral density(BMD), which remain the single best predictor of fracture. In last decade, a large number of genome-wide association analysis(GWASs) and meta-analysis identified several single nucleotide polymorphisms(SNPs) mutation located in the wnt16 gene(7q31)significantly associated with BMD, and it was confirmed in wnt16 knockout mice model. However, the molecular mechanism of wnt16 regulated skeletal development in vivo is unclear, especially in the early period of embryonic development. In our research CRISPR gene editing technology was used to knockout wnt16 gene in zebrafish, and then the effects of Wnt16-/- gene on skeletal development was checked to further reveal the molecular mechanism of bone development.In order to improve the efficiency of target, two target gRNAs were injected into zebrafish embryo at 0-1 cell level, and then target activity was confirmed with T7E1 enzyme and DNA sequencing after 50 hpf(hours post-fertilization). There were 4 mutants of wnt16 gene to be successfully identified in F0 adult zebrafish by TA-clone sequencing. The F0 mutants zebrafish were hybridized with wild type to obtain F1 generation, then 5 different mutations were screened in the F1 generation adult zebrafish. No frameshift mutations were found in Wnt16(21)F1 and wnt16(7)F1, but a part of amino acid sequence changed. Its protein spatial structure was predict by Phyre and found an alpha helix structure changed. The same genotype of males and females zebrafish were hybridized to obtain F2 generation and the females homozygous mutantswere identified in wnt16(21)F2. Cartilage was stained by Alcian blue in Wnt16(21)F2 and wnt16(14)F2 adult zebrafish at 10 dpf and a longer Meckel’s cartilage and a bending spine was found in wnt16(21)F2compared with wild type. These results suggest that wnt16 affect bone development of zebrafish.