Title Page
ABSTRACT
Contents
1. INTRODUCTION 14
2. MATERIALS AND METHODS 20
2.1. Plasmid DNA preparation 20
2.2. Cell culture 21
2.3. HAP1-ABEDox : EGFP reporter cell line generation[이미지참조] 21
2.4. Drug screening 22
2.5. HDAC1 and HDAC2 knockdown cell line generation 23
2.6. Protein purification 24
2.7. Transfection and drug treatment 25
2.8. Targeted deep sequencing and data analysis 26
2.9. Protein expression level measurement 26
2.10. Quantitative PCR 27
2.11. Chromatin immunoprecipitation (ChIP) assay 28
3. RESULTS 29
3.1. Generation of HAP1-ABEDox : EGFP cell line[이미지참조] 29
3.2. Small molecule drug screening to improve adenine base editing efficiency 31
3.3. Romidepsin improves base editing efficiency at endogenous target sites 33
3.4. HDAC inhibitor improves base editing efficiency 35
3.5. Range of base editing window by romidepsin 36
3.6. Inhibition of HDAC1 or HDAC2 expression improves ABE-mediated base editing efficiency 37
3.7. Off-target effect in the absence and presence of romidepsin 38
3.8. Romidepsin increases ABE7.10 protein and gRNA expression level 39
3.9. Romidepsin increases ABE activity by converting the chromatin state. 41
3.10. Romidepsin increases the base editing efficiency of ABEmax and BE4max 43
3.11. Romidepsin increases product purity of base editing 44
4. DISCUSSION 86
5. REFERENCES 91
국문요약 103
Table 1. List of target sequences of gRNA used in the study 81
Table 2. List of primer sequences used in the study 82
Table 3. Top 30 drugs in small molecule drug screening 84
Table 4. List of off-target sites 85
Figure 1. Schematic design of HAP1-ABEDox : EGFP reporter system.[이미지참조] 45
Figure 2. Base editing efficiency of HAP1-ABEDox cell line[이미지참조] 46
Figure 3. Optimization of the HAP1-ABEDox : EGFP reporter cell line.[이미지참조] 47
Figure 4. The relative fold-change of EGFP expression level in drug screens. 50
Figure 5. Adenine base editing efficiency at the EGFP target site of HAP1-ABEDox : EGFP reporter system[이미지참조] 52
Figure 6. Adenine base editing efficiency at endogenous CCR5 target site. 54
Figure 7. Evaluation of toxicity and base editing frequency according to romidepsin concentration 55
Figure 8. Romidepsin improves adenine base editing efficiency at endogenous target sites. 57
Figure 9. Romidepsin improves adenine base editing efficiency in HeLa cell 59
Figure 10. HDAC inhibitors improve adenine base editing efficiency. 60
Figure 11. Effect of base editing window in the absence and presence of romidepsin 63
Figure 12. Inhibition of HDAC1 and HDAC2 expression by romidepsin improves adenine base editing efficiency 64
Figure 13. Analysis of off-target effect by romidepsin. 66
Figure 14. Romidepsin enhances adenine base editor protein expression level 67
Figure 15. Assessment of Cas9- and BE3-mediated mutation frequencies by romidepsin treatment 69
Figure 16. Romidepsin enhances alternative promote-driven ABE7.10 expression level. 71
Figure 17. Romidepsin enhances a U6 promoter-driven gRNA expression level. 73
Figure 18. Romidepsin improves base editing efficiency by affecting the chromatin state. 74
Figure 19. The effect of romidepsin in open and closed chromatin region. 76
Figure 20. Romidepsin improves ABEmax- and BE4max-mediated base editing efficiency. 78
Figure 21. Effect of romidepsin on base editing product purity 80