A collaborative team from the National Tsing Hua University, China Medical University and Chang Gung Memorial Hospital in Taiwan has developed, for the first time, a new bi-directional gene regulation system for the control of stem cell differentiation and tissue regeneration. This work will substantially foster tissue regeneration of patients and benefit stem cell research.
Many tissues in humans, such as articular cartilage and calvarial bone, have limited ability to self-repair. For instance, articular cartilage at the knee degener-ates upon damage, hence losing the ability to provide cushion and resulting in pain at the knee. Mesenchymal stem cell can differentiate into cartilage, bone or fat cell and is an important cell source for tissue repair by regeneration. During differentiation, however, many essential genes are not well regulated, thus leading to poor cell differentiation and tissue regeneration. Precise manipulation of gene expression is crucial to promote tissue repair.
First CRISPR-AI system for bi-directional gene regulation
To manipulate gene expression, Prof. Yu-Chen Hu at National Tsing Hua University and his collaborators lead the world and develop a novel CRISPR-AI bi-directional gene regulation system to simultaneously activate and inhibit different genes in stem cells. The team utilizes a dCas9 protein and designs two single guide RNA (sgRNA) scaffold. The first sgRNA binds with dCas9 and transcription factors to target Sox9 gene that promotes stem cell differentiation to cartilage cell. The second sgRNA binds with dCas9 and transcription repressors and targets to PPAR- gene that directs stem cell differentiation to fat cell.
First application of bi-directional gene regulation system in tissue regenera-tion
To date, there is no precedent to promote tissue regeneration by simultaneous activation and inhibition of multiple genes. Prof Yu-Chen Hu and his team show that delivery of the CRISPR-AI system into stem cells can activate Sox9 for 17-fold and repress PPAR- for 70%, when compared with the original stem cell. By concurrent activation and inhibition of two genes controlling two differentiation paths, the CRISPR-AI system can substantially promote stem cell differentiation towards cartilage and repress differentiation towards fat. Implantation of the engineered cells into bone defects significantly promotes the tissue regeneration. This study paves a new avenue to developing CRISPR-AI for bi-directional gene regulation and tissue repair, and is published in the top international journal “Nucleic Acids Research” in 2019.
CRISPR-AI holds promise for cell therapy and cancer therapy
Prof. Yu-Chen Hu points out that gene regulation is crucial for cells, but bi-directional and precise control of gene expression remains challenging and has yet to be explored for stem cell and tissue regeneration. This CRISPR-AI system can specifically direct the cell differentiation, thus holding promise for stem cell re-search and for repair of cartilage defects or other tissues. A new trend in cancer therapy is the use of immune cells for immunotherapy. CRISPR-AI is also promising to modulate gene expression in immune cells and enhance their ability to kill cancer cells.
Truong, V. A., Hsu, M-N., Nguyen, N.T.K., Lin, M-W., Shen, C.-C., Lin, C.-Y., Hu, Y.-C.* 2019. July. CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration.Nucleic Acids Research. 47: e74 (IF 11.147).
Yu-Chen Hu, PhD
Tsing Hua Chair Professor,
Department of Chemical Engineering,National Tsing Hua University
Dept. of Eng. & Technology, MOST