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Genetic Screens using CRISPR-Cas9 : 크리스퍼 유전자 가위를 이용한 유전자 스크리닝
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- Authors
- Advisor
- 이연
- Major
- 자연과학대학 화학부
- Issue Date
- 2018-08
- Publisher
- 서울대학교 대학원
- Description
- 학위논문 (박사)-- 서울대학교 대학원 : 자연과학대학 화학부, 2018. 8. 이연.
- Abstract
- Human Genome Project (HGP) which launched in 1990 and completed in 2003, revealed the whole sequence of human DNA. After this triumphs, functional genomics, which is a field that attempts to identify which genes contribute to each biological phenotypes including diseases, has attracted much attention based on the rich data given by the HGP and others.
For functional genomics, tools for genetic perturbations are required to connect the phenotypes to genotypes. Especially for forward genetic approaches, which modify expressions of many genes in a time and selecting and characterizing the cells and genes with desired phenotypes, genetic perturbation systems that enable many modifications easily are essential. As a result, programmable nucleases, CRISPR (clustered regularly inter¬spaced short palindromic repeat) – Cas9 system has been spotlighted as a tool for functional genomics because of its simplicity and robustness. Although RNA interference (RNAi) has been major tool for functional genomics, it has been criticized for its drawbacks including high off-target effects and incomplete knock-down. On the other hands, RNA guided Cas9 nuclease has much higher specificity than RNAi system and induce complete knock-out of genes. In this respect, CRISPR-Cas9 system could be an excellent substitute for previous RNAi system in functional genomics.
In this thesis, I will describe two genetic screen approaches that I developed using CRISPR-Cas9 and the result of them. In Chapter 1, I will present new pooled CRISPR screen method using 30,840 single-guide RNAs (sgRNAs) combined with whole genome sequencing to directly identify causal genotype of viral resistance. Compared to screens based on lentivirus integration system, this method is quick, virus-free, and confirms authentic mutations.
Second, in Chapter II, I will describe arrayed CRISPR screen which incorporates a single genetic perturbation in each well, using individually cloned 4,542 sgRNA plasmids targeting 1,514 genes encoding potential host factors for viral infection. Combined with image-based assay detecting viral infection, this arrayed screen could reveal new host factors for coxsackievirus-B3 infection to human cells that are missed in pooled CRISPR screens, demonstrating higher sensitivity of arrayed CRISPR screen.
- Language
- English
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