Optimization of the CRISPR/Cas9 system for excision of genomic regions in human cancer cell cultures

• Main Author: Tng, Jia Qi
• Other Authors: Melissa Jane Fullwood
• Format: Final Year Project
• Language: English
• Published: 2016
• Subjects: DRNTU::Science::Biological sciences::Molecular biology
• Online Access: http://hdl.handle.net/10356/67362
• Institution: Nanyang Technological University

In recent years, the role of chromatin interactions in regulating gene expression has been intensively studied. Although much insight into the factors and mechanisms underlying the establishment and maintenance of chromatin loops has been gained, there still remains much to be understood. In this thesis, somatic mutation hotspots occurring in the noncoding genomic regions identified in Weinhold et al. [Nat Genet, 46(11), 1160-1165, doi:10.1038/ng.3101 (2014)] were shortlisted in human Chronic Myelogenous Leukemia (CML) and Breast cancer disease models K562 and MCF7 following Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET). It is hypothesized that these mutations occur on enhancer/promoter regions, resulting in aberrant gene expression through the dysfunctional regulation of chromatin interactions. Attempts were made to create mutation knockouts in K562 and MCF7 for the study of potential roles these mutations might have in mediating chromatin interactions. EZH2 gene knockouts in the near-haploid CML disease model HAP1 was also performed to test another hypothesis; that off-target activity of the clustered, regularly interspaced, short palindromic repeat (CRISPR)/Cas9 system can induce changes in the chromatin interaction landscape. Optimization of the CRISPR/Cas9 system to K562, MCF7, and HAP1, as well as a suitable screening method for genome-edited clones are described in this thesis.