Title Page
Abstract
Contents
General Introduction 11
1. Significance of genome editing in hPSCs 11
2. Toolbox for precise genome editing in hPSCs 14
2.1. Development of programmable nucleases 15
2.2. Base editors 18
2.3. Prime editors 19
3. Pros and cons of BEs and PE in hPSCs 22
3.1. Gene pencils rather than gene scissor 22
3.2. Limitation of BEs and PE 24
4. Unique cellular characteristic of hPSCs affecting genome editing outcome 26
4.1. High susceptibility to DNA damage stimuli 27
4.2. Active DNA repair systems 28
Chapter 1. Safe scarless cassette free selection of genome edited human pluripotent stem cells using temporary drug resistance 31
I-1. Introduction 32
I-2. Materials and Methods 34
I-3. Results 38
A. YM155-based enriched selection of genome-edited hESCs 38
B. Scarless YES-approach for establishment of CCR5-targeted hESCs 41
C. Scarless YES-approach for gene knockout and knock-in hESCs 48
I-4. Discussion 56
I-5. Graphical summary 59
Chapter 2. High expression of uracil DNA glycosylase determines C to T substitution in human pluripotent stem cells 60
II-1. Introduction 61
II-2. Materials and Methods 64
II-3. Results 68
A. ABE efficiency is distinctively higher than that of CBE in hESCs 68
B. Distinct expression pattern of DNA glycosylases in hPSCs 75
C. Marginal effect of DNA glycosylases on ABE outcomes in hESCs 82
D. UNG expression for efficiency and product purity of CBE in hESCs 85
II-4. Discussion 91
II-5. Graphical summary 94
Chapter 3. MSH2 and MSH6 as size dependent cellular determinants for prime editing in human pluripotent stem cells 95
III-1. Introduction 96
III-2. Materials and Methods 99
III-3. Results 101
A. High enrichment of MMR pathway in hPSCs 101
B. Distinctive expression levels of MSH2 and MSH6 genes in hPSCs 103
C. Genetic perturbation of MSH2 or MSH6 in hESCs with CRISPR/Cas9 106
D. Discrimination of editing size of PE by MSH2 or MSH6 110
III-4. Discussion 114
III-5. Graphical summary 116
Chapter 4. Optimized base and prime editor for human pluripotent stem cells via dual inhibition of DNA damage response and DNA repair pathway 117
IV-1. Introduction 118
IV-2. Materials and Methods 121
IV-3. Results 124
A. Additive effect of dual inhibition of the DNA damage response and DNA repair pathway 124
B. Optimized cytosine base editor for human pluripotent stem cells via dual inhibition of DNA repair and DNA damage response 129
C. Marginal effect of p53DD in nutlin-3 resistant hiPSCs 132
D. Optimized prime editor for human pluripotent stem cells via dual inhibition of DNA repair and DNA damage response 136
E. p53DD reduces the enrichment of TP53 mutant population during genome editing 139
IV-4. Discussion 143
Chapter 5. Multiple isogenic GNE myopathy modeling with mutation specific phenotypes from human pluripotent stem cells by base editors 145
V-1. Introduction 146
V-2. Material and Method 149
V-3. Results 155
A. Target coverage of point mutations causing GNE myopathy by base editors 155
B. Efficiency of base editing in hESCs 158
C. Establishment of GNE myopathy disease models through base editors 163
D. Mutation-specific phenotype of GNE myopathy disease models 168
E. Structural analysis predicts mutation-specific hyposialylation 176
F. Mutation-specific drug response of GNE myopathy disease models 180
G. GNE myopathy modeling in myocytes derived from GNE-hESCs 184
V-4. Discussion 187
V-5. Graphical summary 190
Reference 195
국문 초록 206
Table 1. PCR primer sequences 191
Table 2. sgRNA sequences 192
Table 3. pegRNA sequences 193
Table 4. qPCR primer 194
Figure 1. Application of human pluripotent stem cells for disease modeling and cell therapy 13
Figure 2. Programmable genome editing tools. 17
Figure 3. Development of BEs and PE 20
Figure 4. Large on-target defect by DSB 23
Figure 5. Limitation of BEs and PE 25
Figure 6. Unique cellular characteristic of hPSCs affecting genome editing outcome 30
Figure 7. YM155 based enriched selection of genome-edited hESCs 40
Figure 8. Scarless YES-approach for establishment of CCR5 targeted hESCs 45
Figure 9. Characterization of CCR5 targeted hESC clone 47
Figure 10. Scarless YES-approach for multiple knockout targets 51
Figure 11. Scarless YES-approach for EGFP knockout 52
Figure 12. Scarless YES-approach for multiple knock-in targets 54
Figure 13. Scarless YES-approach for EYFP knock-in targets 55
Figure 14. ABE-mediated editing efficiency is distinctively higher than that of CBE in hESCs 71
Figure 15. Effect of p53 in base editing efficiency 74
Figure 16. Highly activated base excision repair in hPSCs 79
Figure 17. Distinct expression pattern of DNA glycosylases in hPSCs 81
Figure 18. Marginal effect of DNA glycosylases on ABE outcomes in hESCs 84
Figure 19. UNG expression for efficiency and product purity of CBE in hESCs 89
Figure 20. Highly enriched mismatch repair pathway in hPSCs 102
Figure 21. High expression of MSH2 and MSH6 in hPSCs 105
Figure 22. Genetic perturbation of MSH2 or MSH6 in hESCs with CRISPR/Cas9 109
Figure 23. Discrimination of editing size of PE by MSH2 or MSH6 113
Figure 24. Additive effect of dual inhibition of the DNA damage response and DNA repair pathway 127
Figure 25. Optimized cytosine base editor for human pluripotent stem cells via dual inhibition of DNA repair and DNA damage response 131
Figure 26. Marginal effect of p53DD in iPSC with resistance to Nutlin-3 134
Figure 27. Optimized prime editor for human pluripotent stem cells via dual inhibition of DNA repair and DNA damage response 138
Figure 28. Reduced enrichment of hESCs harboring mutations in TP53 gene by p53DD during cytosine and prime editing 142
Figure 29. Target coverage of point mutations causing GNE myopathy by base editors 157
Figure 30. Efficiency of base editing compared to HDR in hESCs 161
Figure 31. Establishment of GNE myopathy disease models through base editors 165
Figure 32. Establishment of GNE myopathy disease models through base editors in multiple hPSCs 167
Figure 33. Mutation-specific phenotype of GNE myopathy disease models 172
Figure 34. Mutation-specific phenotype of GNE myopathy disease models from multiple hPSCs 174
Figure 35. Distinctive basal sialic acid levels in multiple hPSCs 175
Figure 36. Structural analysis predicts mutation-specific hyposialylation 179
Figure 37. Graphical description of biosynthesis of sialic acid and sialyation and roles of GNE protein as UDP-GlcNAc-2-epimerase and ManNAc kinase 181
Figure 38. Mutation-specific drug response of GNE myopathy disease models 183
Figure 39. GNE myopathy modeling in myocytes derived from GNE-hESCs 186