A genome-wide library of TAL effector nucleases

학위논문 (박사)-- 서울대학교 대학원 : 화학부(생화학전공), 2014. 2. 김진수.Transcription activator–like (TAL) effector nucleases (TALENs) are newly developed programmable nucleases which use a simple protein–DNA code that relates modular DNA-binding TALE repeat domains to individual bases in a target binding site. Unlike homing endonucleases and zinc figer nucleases (ZFNs), they can be readily engineered to bind specific genomic loci, enabling the introduction of precise genetic modifications such as gene disruptions, additions, corrections and genome rearrangements. In this thesis, I developed improved TALEN architectures to avoid unwanted mutations in genome. Then, I carefully chose genome-wide TALEN target sites that did not have highly similar sequences elsewhere in the genome and assembled TALEN pairs using high throughput Golden Gate cloning system. A pilot test including over a hundred pairs of TALENs showed that all TALENs were active and disrupted their target genes at high frequencies, although two of these TALENs became active only after their target sites were partially demethylated using a DNA methyltransferase inhibitor. I used the TALEN library to generate single- and double-gene knockout cells in which NF-kB signaling pathways were disrupted. Compared with cells treated with short interfering RNAs (siRNA), these cells showed unambiguous suppression of the signal transduction. Furthermore, I developed the TALEN library for targeting every exon in protein coding genes of several organisms including human. The TALEN library reported here will be broadly useful for research and drug discovery.Abstract i Table of Contents iii List of Figures v List of Tables viii List of Abbreviations ix I. Introduction 1 II. Materials and Methods 1. Dual fluorescent reporter plasmid construction 4 2. Cell culture and transfection 4 3. High-throughput assembly of TALENs 4 4. Flow cytometry 6 5. T7E1 assay for mutation detection 6 6. PCR analysis to detect genomic mutations and sequencing 7 7. Analysis and rescue of genome-inactive TALENs 8 8. Digital PCR to estimate mutation frequency 8 9. Gene-knockout cell lines 8 10. Fluorescent PCR analysis 9 11. Episomal reporter assay to detect NF-κB signaling 9 12. Western blotting 10 III. Results A. Optimization of TAL Effector Nucleases (TALENs) 1. Dual fluorescent reporter system 11 2. Design of prototype TALENs 15 B. Human genome-wide TALEN library 1. One-step Golden-Gate cloning system 18 2. Design of human genome-wide TALENs 29 3. TALEN-mediated genome editing activity 36 4. Rescue of inactive TALENs 46 5. Undetectable off-target mutations 52 6. TALEN induced genome rearrangement 56 C. TALEN-mediated knockout cell lines 1. Establishment of knockout cell lines for NF-κB pathway study 63 2. Episomal reporter assay 73 D. Expansion of TALEN library 1. Design of expanded TALEN library 84 2. TALEN library for several organisms 91 E. Comparison of ZFNs and TALENs 96 IV. Discussion 105 V. Appendix 1. List of TALEN activity in reporter assay 110 2. List of TALEN activity in the T7E1 assay 120 VI. References 123 Abstract in Korean 136Docto