A genome-wide library of TAL effector nucleases
- 자연과학대학 화학부
- Issue Date
- 서울대학교 대학원
- 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.