Computational analysis of DNA and RNA editing in human cells

• Main Author: Srinivasan, Harini
• Other Authors: Tan Meng How
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Engineering::Chemical engineering
• Online Access: https://hdl.handle.net/10356/136576
• Institution: Nanyang Technological University

Use of computational techniques is inevitable in present day biological research and this work describes my efforts in utilizing these computational techniques on biological systems in combination with experiments to increase our understanding of them. DNA editing (using CRISPR-Cas system) and RNA editing (ADAR-mediated) in human cells are studied in this work with an aim to increase our understanding of functioning of these editing mechanisms. CRISPR-Cas systems are RNA-guided DNA editing systems that have their origin in bacteria and archaea. They hold huge potential for genome engineering due to their ability to introduce a double-stranded break at any desired genomic location. In an attempt to increase the understanding of the functioning of these CRISPR-Cas editing systems, a structural study was designed to probe the role of conserved residues in the active site of the HNH nuclease domain of Cas9. This study highlighted the likely role of highly conserved residues in maintaining the structure of the active site cleft in the HNH nuclease domain and thereby, in the functioning of the enzyme. Succeeding these efforts, another study based on activity of enzymes was designed to gain insights into the functioning of the CRISPR-Cas enzymes. Here a large scale characterization of the functioning of five CRISPR-Cas enzymes was done by comparing their performance in genome editing pathways using a combination of experiments and computational pipelines developed to analyze and quantify the NGS data from the experiments. This study highlighted the factors affecting the functioning of the five enzymes and their performances. Additionally, the mutations introduced by the five enzymes in NHEJ-mediated genome editing was identified to reveal non-random patterns in the next study. This study enabled characterizing the most probable insertion and deletion outcomes from the NHEJ-mediated genome editing using the five enzymes. The results from both these studies give us insights that can serve as a guidance for designing future genome engineering studies and also enable using the CRISPR-Cas technology safely to its fullest potential. Further, an application of CRISPR-Cas editing system was showcased by using it to produce a glutamine synthase knockout cells based on HEK293 cells in the next study. These knockout cells were characterized for their utility as a bioproduction platform for stable production of glycoproteins with resemblance to human proteins. These cells are then developed into a suitable expression system for stable production of recombinant erythropoietin (EPO) with fully human glycosylation. Finally, another aspect of gaining insights into the functioning of a system involves understanding its regulators and RNA editing system was employed for this study. RNA editing refers to site-specific nucleotide change in RNA after transcription, and adenosine to inosine (A-to-I) editing is the most prevalent modification in higher eukaryotes mediated by ADAR family of enzymes. In this study, regulators of ADAR-mediated RNA editing were probed for using a combination of experiments and genome-wide computational study with an objective of gaining insights into its mechanism. This study revealed splicing factor, SRSF9, as a key repressor of ADAR2-mediated editing of brain-specific sites in non-brain tissues, thereby, enabling the shaping of the editing landscape.