Utilizing CRISPR/Cas9 to Study SOX9 Haploinsufficiency in Human iPSC-derived Chondrocytes
Refereed conference paper presented and published in conference proceedings



摘要SOX9 is an important gene in development which actively involves in the differentiation of 3 germ layers and it is the master regulatory gene in chondrocyte development. Moreover, haploinsufficiency (HI) in SOX9 will result in severe developmental disorder called Campomelic Dysplasia (CD). Patients with CD suffer from bent long bones, club feet, small thoracic cavity etc. CD patients usually die shortly after birth due to respiratory failure and only very few of them could survive into adolescent years. Our study aims at elucidating the effect of different pathways which are important for SOX9 HI on chondrocytes.
To generate our desired SOX9 HI model, we utilized CRISPR/Cas9 to make a single nucleotide polymorphism (SNP) mutation at the end of SOX9 exon 2 splice site in human induced pluripotent stem cells (hiPSCs). Single clones were screened by Sanger sequencing of the PCR fragments. The SOX9 HI hiPSCs were differentiated to chondrocytes according to a well-established 14-day protocol. To validate our desired SOX9 HI mutant, RT-qPCR, immunofluorescence and Safranin O staining were performed on day 14. Our results from these experiment consistently showed that the SOX9 HI chondrocytes have decreased expression of SOX9 in terms of mRNA and protein levels.
Transcriptome analysis were then performed. By comparing wild type chondrocytes and SOX9 HI chondrocytes, we identified around 700 differentially expressed genes for each phenotype and from gene ontology analysis, binding of around 1000 genes were being affected in SOX9 HI chondrocytes. From the KEGG analysis results, notch signaling, p53 and ribosomal pathways have the highest rich factor scores.
To conclude, we had successfully generated SOX9 HI hiPSCs and differentiated them to chondrocytes. From the transcriptome analysis results, several pathways were being affected in SOX9 HI chondrocytes. Our further research would be to inhibit these pathways one by one and try to rescue the SOX9 HI phenotype with different approaches.
著者Tin Yan HA, Kelvin Kai Kei MIU, Wai Yee CHAN
會議名稱ASHG 2019 Annual Meeting

上次更新時間 2020-02-06 於 09:53