Laboratory of Nano-regenerative Medicine, Department of Biomedical Science, College of Life Science, CHA University, CHA Biocomplex, Sampyeong-Dong, Bundang-gu, Seongnam-si, 13488, Republic of Korea.
Molecular Genetics, Department of Biomedical Science, College of Life Science, CHA University, 605, CHA Biocomplex, Sampyeong-Dong, Bundang-gu, Seongnam-si, 13488, Republic of Korea.
Biomater Sci. 2022 Jan 18;10(2):514-523. doi: 10.1039/d1bm01716k.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system recognizes and deletes specific nucleotide sequences in cells for gene editing. This study aimed to edit and knockdown the RUNX2 gene, a key transcription factor that is directly involved in all stages of stem cell differentiation into osteoblasts. The RUNX2 gene was depleted using the CRISPR-Cas9 system to inhibit osteoblast differentiation of stem cells. shRNA vectors targeting RUNX2 were used as a control. The surface of nanoparticles (NPs) was coated with the cationic polymer linear polyethyleneimine. Thereafter, negatively charged CRISPR-Cas9 and shRNA vectors were complexed with positively charged NPs ionic interactions. Several analytical methods were used to determine the size, surface charge, and morphology of NPs and to characterize the complexed genes. NPs complexed with CRISPR-Cas9 and shRNA vectors were delivered into human mesenchymal stem cells (hMSCs) endocytosis. The mRNA and protein expression patterns of various genes in hMSCs were measured over time following internalization of NPs complexed with CRISPR-Cas9 and shRNA vectors in two- and three-dimensional culture systems. Knockdown of the RUNX2 gene decreased osteogenic differentiation and increased chondrogenic differentiation of hMSCs. As a result of investigating the efficiency of NPs complexed with CRISPR-Cas9 (CASP-NPs), Runx2 effectively knocked down in mesenchymal stem cells to enhance differentiation into chondrocytes, therefore CASP-NPs proved to be an effective gene carrier in hMSCs.
簇状规律间隔短回文重复序列 (CRISPR)-Cas9 系统可识别和删除细胞内特定的核苷酸序列，用于基因编辑。本研究旨在编辑和敲低 RUNX2 基因，该基因是直接参与干细胞向成骨细胞分化的所有阶段的关键转录因子。CRISPR-Cas9 系统用于耗尽 RUNX2 基因，以抑制干细胞的成骨细胞分化。使用靶向 RUNX2 的 shRNA 载体作为对照。纳米颗粒 (NPs) 的表面涂有阳离子聚合物线性聚乙烯亚胺。此后，带负电荷的 CRISPR-Cas9 和 shRNA 载体与带正电荷的 NPs 通过离子相互作用复合。使用几种分析方法来确定 NPs 的大小、表面电荷和形态，并对复合基因进行表征。CRISPR-Cas9 和 shRNA 载体复合的 NPs 通过内吞作用进入人骨髓间充质干细胞 (hMSC)。在二维和三维培养系统中，测量 hMSCs 内化 CRISPR-Cas9 和 shRNA 载体复合的 NPs 后，随时间推移各种基因的 mRNA 和蛋白表达模式。RUNX2 基因的敲低降低了 hMSC 的成骨分化，增加了软骨分化。通过研究与 CRISPR-Cas9 复合的 NPs (CASP-NPs) 的效率，Runx2 在间充质干细胞中有效敲低以增强向软骨细胞的分化，因此 CASP-NPs 被证明是 hMSCs 中的有效基因载体。
