生命科学学院学术报告
题目：Genome editing using CRISPR-guided cytidine deaminase
主讲人：常兴
中国科学院上海生命科学研究所
时间：11月9日（星期五）14：00-15：30
地点：王克桢楼348会议室
联系人：茅傲岳（本科生，18801382265）；李毓龙研究员
摘要：
Genomic DNA not only stores coding information of proteins, but also encodes critical regulatory information for proper gene expression, including gene transcription, RNA splicing and translation. Understanding normal physiology and treatment of many human diseases thus requires precise manipulation of genomic DNA. CRISPR-guided cytosine deaminase can efficiently convert targeted C to T or G to A in the presence of a uracil glycosylase inhibitor (UGI), enabling efficient base editing in mammalian cells. We show that CRISPR-guided cytosine deaminase (i.e. Target-AID induced mutagenesis, TAM) can be harnessed to treat human diseases beyond correcting point mutations, including optimizing monoclonal antibodies and correcting splicing defects for inherited diseases. 
First, TAM directly subverts cytidines or guanines to the other three bases, generating a large repertoire of protein variants for protein evolution. We show that combining TAM with mammalian surface display is able to randomly mutate the CDR regions of immunoglobulin, allowing screening for higher-affinity antibodies in vitro. Second, we show that directing TAM to critical cis-elements is able to modulate various forms of mRNA alternative splicing at their native expression level. We further demonstrate that this strategy was readily adoptable to unravel novel functions of splicing isoforms and to correct splicing defects associated with human diseases. 
In summary, our results demonstrate that the targeted cytosine deaminase provides an incisive new tool to increase our understanding of biology and to discover treatments for human disease.
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