BIOPIC学术报告
报告题目：CRISPR-Cas9 Application in Mouse Model Creation, T cell engineering, and Transcription Regulation
报告人：王皓毅  研究员
中国科学院动物研究所
时间：2016年12月13日上午10:30
地点：英国威廉希尔公司综合科研楼302会议室
联系人：何姣，62767408，hejiao@pku.edu.cn
Abstract
CRISPR-Cas9 system has become the tool of choice for genome engineering. Previously we established the method of microinjecting CRISPR-Cas9 system into zygotes to generate mouse models. To overcome the technically demanding and inherently low throughput method of microinjection, we devised the Zygote Electroporation of Nuclease (ZEN) technology, which employs electroporation to deliver CRISPR-Cas9 reagents to the zygotes and generated live mice carrying targeted NHEJ and HDR mutations with high efficiency. The general principles discovered and described in this study have implications for high efficiency, high throughput genome engineering in animals.
We applied CRISPR-Cas9 mediated gene editing to chimeric antigen receptor (CAR) T cells. We disrupted multiple genes—β2 microglobulin (B2M), TCRα constant (TRAC), and programmed death-1 (PD1; CD279)—and successfully generated double (TRAC, B2M) and triple (TRAC, B2M, and PD-1) negative CAR-T cells. These edited CAR-T cells lost the expression of TCR and HLA class I molecules on the cell surface and retained potent anti-tumor capability in an antigen-specific manner. We show that CRISPR-Cas9 mediated multiplex gene editing is a promising method to generate universal CAR-T cells from allogeneic healthy donors.
To extend the utility of the CRISPR-Cas9 system, we have taken advantage of the ability of Pumilio PUF domains to bind specific 8-mer RNA sequences. By combining these two systems, we established the Casilio system, which allows for specific and independent delivery of effector proteins to specific genomic loci. We demonstrated that the Casilio system enables independent up- and down-regulation of multiple genes, as well as live-cell imaging of multiple genomic loci simultaneously. Importantly, multiple copy of PUF binding sites can be incorporated on sgRNA backbone, therefore allowing for local multimerization of effectors. In addition, the PUF domain can be engineered to recognize any 8-mer RNA sequence, therefore enabling the generation and simultaneous operation of many Casilio modules.
Key Words: Genome editing, CRISPR-Cas9, mouse model, CAR-T, transcription regulation
Brief  Introduction of Haoyi Wang, 王皓毅
Dr. Wang has an interdisciplinary training in genetics, molecular biology, and stem cell biology. As a doctoral and post-doctoral researcher, Dr. Wang has worked on the development of a variety of genome engineering technologies, including transposon-based “Calling Card” method for determining the genome-wide binding locations of transcription factors, TALEN-mediated genome editing in human pluripotent stem cells and mice, CRISPR-mediated multiplexed genome editing in mice, and CRISPR-mediated gene activation in human cells.
In 2014, he established his own lab at the Institute of Zoology, Chinese Academy of Sciences (Beijing, China). Since then, Wang lab has developed Zygote Electroporation of Nuclease (ZEN) method to generate genetically modified mouse models with high throughput and efficiency, Casilio method to regulate gene transcription, as well as method to generate CAR-T cells with multiplex gene editing. As the Distinguished Visiting Professor at the Jackson Laboratory, Dr. Wang directs a small but focused research team and work closely with the Genetic Engineering Technology group, to improve the efficiency and capability of generating genetically modified mouse models.
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