CRISPR/Cas9 technology in disease research and therapy: a review / 生物工程学报

Genome editing is a genetic manipulation technique that can modify DNA sequences at the genome level, including insertion, knockout, replacement and point mutation of specific DNA fragments. The ultimate principle of genome editing technology relying on engineered nucleases is to generate double-stranded DNA breaks at specific locations in genome and then repair them through non-homologous end joining or homologous recombination. With the intensive study of these nucleases, genome editing technology develops rapidly. The most used nucleases include meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats associated Cas proteins. Based on introducing the development and principles of above mentioned genome editing technologies, we review the research progress of CRISPR/Cas9 system in the application fields of identification of gene function, establishment of disease model, gene therapy, immunotherapy and its prospect.

An update on the tools for creating transgenic animal models of human diseases - focus on atherosclerosis

Animal models of diseases are invaluable tools of modern medicine. More than forty years have passed since the first successful experiments and the spectrum of available models, as well as the list of methods for creating them, have expanded dramatically. The major step forward in creating specific disease models was the development of gene editing techniques, which allowed for targeted modification of the animal's genome. In this review, we discuss the available tools for creating transgenic animal models, such as transgenesis methods, recombinases, and nucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems. We then focus specifically on the models of atherosclerosis, especially mouse models that greatly contributed to improving our understanding of the disease pathogenesis and we outline their characteristics and limitations.

Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology

Recent developments in genome editing technology using meganucleases demonstrate an efficient method of producing gene edited pigs. In this study, we examined the effectiveness of the transcription activator-like effector nuclease (TALEN) system in generating specific mutations on the pig genome. Specific TALEN was designed to induce a double-strand break on exon 9 of the porcine α1,3-galactosyltransferase (GGTA1) gene as it is the main cause of hyperacute rejection after xenotransplantation. Human decay-accelerating factor (hDAF) gene, which can produce a complement inhibitor to protect cells from complement attack after xenotransplantation, was also integrated into the genome simultaneously. Plasmids coding for the TALEN pair and hDAF gene were transfected into porcine cells by electroporation to disrupt the porcine GGTA1 gene and express hDAF. The transfected cells were then sorted using a biotin-labeled IB4 lectin attached to magnetic beads to obtain GGTA1 deficient cells. As a result, we established GGTA1 knockout (KO) cell lines with biallelic modification (35.0%) and GGTA1 KO cell lines expressing hDAF (13.0%). When these cells were used for somatic cell nuclear transfer, we successfully obtained live GGTA1 KO pigs expressing hDAF. Our results demonstrate that TALEN-mediated genome editing is efficient and can be successfully used to generate gene edited pigs.

Applications of ZFN, TALEN and CRISPR/Cas9 techniques in disease modeling and gene therapy / 中华医学遗传学杂志

Precise and effective modification of complex genomes at the predicted loci has long been an important goal for scientists. However, conventional techniques for manipulating genomes in diverse organisms and cells have lagged behind the rapid advance in genomic studies. Such genome engineering tools have featured low efficiency and off-targeting. The newly developed custom-designed nucleases, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system have conferred genome modification with ease of customization, flexibility and high efficiency, which may impact biological research and studies on pathogenesis of human diseases. These novel techniques can edit the genomic DNA with high efficiency and specificity in a rich variety of organisms and cell types including the induced pluripotent stem cells (iPSCs), which has conferred them with the potential for revealing the pathogenesis and treatment of many human diseases. This review has briefly introduced the mechanisms of ZFN, TALENs and CRISPR/Cas9 system, and compared the efficiency and specificity of such approaches. In addition, the application of ZFN, TALENs and CRISPR/Cas9 mediated genome modification for human disease modeling and gene therapy was also discussed.