A CRISPR-engineered Swine Model Of COL2A1 Deficiency Recapitulates Altered Early Skeletal Developmental Defects In Humans

Research Background:Genetic skeletal Disorders are a series of skeletal dysplasias caused by mutations in genes that regulate skeletal and cartilage development.Previous studies have found that the COL2A1 gene encoding type II collagen is an important gene regulating cartilage development,and mutations at different sites on the COL2A1 gene can cause a series of phenotypes of orthopedic diseases.Moreover,the range of COL2A1-related clinical severity is very large.So far,nevertheless,the pathological mechanism of COL2A1 mutation is not under clear conclusions.CRISPR / Cas9 is a new gene-editing tool developed in 2013.Under the guidance of gRNA,the Cas9 endonuclease can accurately find the target site and efficiently cut double-stranded DNA.This tool provides a solid basis for the study of gene mutations.In recent years,genetically modified pig models have played a key role in scientific research in the fields of biology,medicine,and agriculture.By using methods of genetic modification,pig models can be established as models of human diseases and donors of heterogeneous organs to contribute to human health.In this experiment,the aim was to build pig models carrying the COL2A1 mutation to further study the phenotype and pathogenic mechanisms of COL2A1 mutant pigs.Research method:1.Multiple pairs of gRNAs targeting exon 27 of the COL2A1 gene were designed and plasmids containing the gRNA and Cas9 protein were constructed.2.By collecting pig embryos and performing cell isolation and culture,pig embryo fibroblasts that meet the transfection conditions were obtained.3.The gRNA/Cas9 recombinant plasmids were transferred to pig embryo fibroblasts by electro-transfection,respectively,and the most efficient gRNA/Cas9 recombinant plasmid was selected by DNA sequencing.4.The recombinant plasmid and the DNA template were co-transfected into pig embryo fibroblasts by electro-transfection,and the screening and identification methods were used to obtain monoclonal cells containing the target mutant fragment.5.Somatic cell nuclear transfer technology and surgical methods were used to place the recombinant embryos into surrogate pigs.6.The genome of cloned piglets and potential off-target sites were identified after the birth of piglets.7.Radiographic imaging,transcriptomics and proteomics analysis of cloned piglets were used to determine the pathological mechanism of COL2A1 gene mutation.Research result:Through monoclonal identification,out of 366 electro-transfected pig embryo fibroblasts,7 of the monoclonal cells had mutations in exon 27 of the COL2A1 were selected.Finally,a single clone with a compound heterozygous mutation was selected and underwent somatic cell nuclear transfer.The genotype of the piglets was the same as that of the monoclonal cell and the COL2A1 region of paired chromosome 13 includes c.1744G>A and c.1749 delA mutations,respectively.Moreover,no off-target phenotype was identified.The transgenic piglets showed severe skeletal deformity,short and soft limbs,flattened nose on the face,and lost vital signs shortly after birth.Then the piglet model was dissected and it was found that the cartilage of its limbs had no normal ossification and joint hypoplasia,the ribs were significantly shortened and the thorax was smaller.Histological examination revealed that the cartilage tissue structure of piglets exhibited a large number of vacuole structures,and saffron O/fast green staining showed fewer cartilage components in cartilage tissue.Further investigation of the abnormal development of the piglets revealed that there was no significant difference in the organs except the lung.The mutant lung is dramatically smaller than the control,and the trachea cartilage of the COL2A1 mutant pig model collapsed.Histological analysis revealed that the tracheal cartilage of the mutant pig model did not have normal support functions,which was the cause of the trachea collapse.Conclusion:1.This experiment successfully constructed a pig model carrying the COL2A1 mutation,and this model showed a significant skeletal abnormal development phenotype,which provided a basis for exploring the pathogenic mechanism of the COL2A1 mutation.2.Analysis of histology,transcriptomics,and proteomics revealed that the expression of type II collagen in articular cartilage and tracheal cartilage tissues was significantly reduced.3.The collapse of tracheal cartilage was found in piglets.It was concluded that the collapse of tracheal cartilage was an important factor leading to the death of piglets through histology and immunohistochemical examination.To sum up,this study is the first time to construct a large mammalian model carrying human genetic and skeletal diseases.This model is helpful to explore human genetic and skeletal diseases and expands the application of gene-editing technology in large mammalian models.