Establishment of Chinese Hamster Ovary Cell Line Development and Engineering Platforms Using CRISPR/Cas9 and Recombinase-mediated Cassette Exchange
- 주제(키워드) Chinese hamster ovary , CRISPR , RMCE , Site-specific integration , Cell engineering
- 주제(DDC) 547
- 발행기관 아주대학교 일반대학원
- 지도교수 Jae Seong Lee
- 발행년도 2024
- 학위수여년월 2024. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000034163
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Developing recombinant Chinese hamster ovary (CHO) cell lines via random integration (RI) is a labor-intensive and time-consuming process, limiting the large- scale production of biopharmaceuticals. Site-specific integration (SSI) is gaining attention as a next-generation method for cell line development (CLD). However, the application of SSI is constrained by low homology-directed repair pathway in CHO cells. Therefore, improving SSI efficiency is crucial for achieving streamlined and predictable CHO CLD. In this study, I established efficient SSI strategies for the (multiple) integration of transgene(s) using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The biopharmaceutical market is rapidly growing, particularly with the advent of novel, but complex therapeutics such as bispecific antibodies. These proteins are often difficult-to-express (DTE) using current CLD methods because they can induce stress in the protein synthesis and secretion pathways, leading to apoptosis. To address these intracellular bottlenecks, substantial cell engineering is necessary to develop advanced CHO factories. Successful mammalian cell engineering requires precise control over gene expression in both quantitative and timely manner, depending on target genes. However, traditional methods, including transient expression or RI, lack precision and sustainability. Therefore, the development of reliable platforms that overcome these limitations is highly valuable in the field of mammalian synthetic biology. In this study, I developed a streamlined doxycycline-inducible CLD platform that enables precise and sustainable control of various effector genes through CRISPR/Cas9 and recombinase-mediated cassette exchange (RMCE). Case studies highlighting the effectiveness and applicability of this platform are presented. Another critical aspect of successful mammalian cell engineering is the appropriate engineering target selection depending on the specific contexts (e.g. expression host, therapeutic proteins). However, targets identified through knowledge- or omics/prediction-based approaches, may not always be reproducible and effective, due to the incomplete existing data and varying engineering contexts. In this study, I conducted RMCE-based genome-scale CRISPR knockout (KO) screening in two CHO cell lines to understand genetic functions and unveil novel targets. By comparing guide RNA distributions in pooled libraries from next- generation sequencing (NGS) data, I identified core and conditional essential genes, as well as candidate screen hits responsible for efficient biomanufacturing. I demonstrated the robustness and reproducibility of the obtained NGS data through technical validations and target gene validation, highlighting their potential use in diverse functional genomics applications, such as DTE protein production. Collectively, the established CHO CLD and engineering platforms using CRISPR/Cas9 and RMCE which include multiple SSI, reliable gene expression control, and genome-wide KO/functional genomics would contribute to improve productivity of recombinant cell lines and accelerate candidate drug screening and large-scale production, addressing the increasing unmet medical needs.
more초록/요약
무작위적 유전자 삽입을 사용한 재조합 동물세포주 개발은 많은 시간과 노동이 필요하며, 바이오 의약품의 대량생산에 한계를 있음을 보여주었다. 이에 대한 대안으로, 위치 특이적 유전자 삽입을 기반으로 한 세포주 개발이 차세대 방법으로 주목받고 있지만, 초세포에서의 낮은 상동 재조합 효율로 인해 실제 적용에 여러 제약이 따랐다. 따라서, 위치 특이적 유전자 삽입 효율을 개선함으로써 효율적이고 예측 가능한 초세포 주 개발이 가능할 것으로 기대된다. 본 연구에서는 clustered regularly interspaced short palindromic repeats (CRISR)/CRISPR-associated protein 9 (Cas9)을 기반으로 한 (다중) 위치 특이적 외래유전자 삽입 기술을 확립하였다. 빠르게 성장하는 바이오 의약품 시장에서, 이중 특이항체와 같은 구조가 점점 더 복잡한 후보 신약들이 등장하고 있다. 이러한 단백질들은 기존의 플랫폼을 사용하여 생산하기 어려운 난발현 단백질로 분류되며, 그 특성에 따라 단백질 합성 및 분비 경로에서 스트레스를 유발하고, 세포 사멸 초래하는 것으로 알려져 있다. 이러한 세포 내부에서 발생하는 문제를 해결하고 차세대 세포주 개발을 위해 세포엔지니어링이 절대적으로 필요하다. 효과적인 동물 세포엔지니어링은 유전자의 특성에 따라 발현량과 시기를 정확하게 제어함으로써 구현할 수 있다. 그러나 일시발현이나 무작위 유전자 삽입을 통한 기존의 동물 세포엔지니어링은 정교하지 못하며 지속 가능성이 떨어지는 문제에 직면해 있다. 따라서 이러한 단점을 극복하고 동물 세포 합성 생물학 연구에 사용될 수 있는 플랫폼 기술로서, 신뢰할 수 있는 유도발현 세포주의 개발이 큰 가치가 있을 것으로 기대된다. 본 연구에서는 CRISPR/Cas9과 재조합 효소 매개 카세트 교환 방법 (RMCE)을 통해 다양한 표적 유전자의 발현을 정교하고 지속 가능하게 제어할 수 있는 독시사이클린 유도 발현 세포주 개발법을 확립하였다. 또한 사례 연구를 통해 그 유용성과 가치를 입증하였다. 효과적인 동물세포 엔지니어링의 또 다른 중요한 요소는 환경과 맥락에 따른 적절한 표적 단백질을 선별하는 것이다. 하지만 이전에 지식 또는 오믹스/예측에 기반하여 효과를 보였던 표적 단백질들은 불완전한 데이터와 맥락에 따라 재현성과 효과가 제한적일수 있다. 따라서 본 연구에서는 두 초세포주에서 RMCE 매개 전장 CRISPR 녹아웃 스크리닝을 수행하여, 유전자의 기능을 이해하고 신규 타겟을 발굴하고자 하였다. 결과적으로, 차세대 염기서열 분석을 통해 얻은 데이터를 활용하여 라이브러리 내 가이드 RNA 분포를 상호 비교하였으며, 핵심 또는 조건에 따른 필수유전자 및 바이오 의약품 생산에 관한 이로운 표현형을 보이는 표적 유전자들을 선별하였다. 해당 기술을 통해 얻어낸 데이터는 신뢰 및 재현 가능하며, 난발현 단백질 생산과 같은 다양한 표현형에 부합하는 새로운 타겟을 발굴하는데 활용될 수 있음을 검증하였다. 본 연구에서 CRISPR과 RMCE기술로 개발된 (다중) 위치 특이적 유전자 삽입, 신뢰 가능한 유전자 발현조절, 그리고 전장 유전체 녹아웃/기능 유전체학 기반의 엔지니어링 플랫폼은 재조합 초세포주의 생산성을 증대하고 후보 신약의 검증 및 대량 생산 기간을 혁신적으로 단축시켜, 향후 증가할 의료수요 충족에 기여할 것으로 기대된다.
more목차
CHAPTER 1. General introduction 1
1.1. Chinese hamster ovary (CHO) cell line development and engineering 2
1.1.1. From random to site-specific integration (SSI) 2
1.1.2. CRISPR/Cas9 and recombinase-mediated cassette exchange (RMCE) 3
1.2. DNA double-strand breaks (DSBs) repair pathway 6
1.2.1. HDR-mediated SSI 6
1.2.2. Strategies for enhancing SSI efficiency 6
1.3. Toward successful CHO cell engineering 13
1.3.1. Mammalian cell engineering platform with GOF mutation 13
1.3.2. Genome-wide CRISPR KO screening 13
CHAPTER 2. Improving the CRISPR/Cas9-mediated site-specific integration efficiency in CHO cells 16
2.1. Abstract 17
2.2. Introduction 18
2.3. Materials and Methods 20
2.3.1. Molecular cloning for plasmid construction 20
2.3.2. Construction of KI monitoring cell lines 21
2.3.3. Cell culture and transfection 22
2.3.4. Evaluation of KI efficiency by flow cytometry 23
2.3.5. Genomic DNA extraction and junction PCR 23
2.3.6. Cell cycle synchronization 24
2.3.7. Cell cycle analysis 24
2.3.8. Statistical analysis 25
2.4. Results 35
2.4.1. Establishment of promoter trap-based KI monitoring cell line 35
2.4.2. Limited effects of conventional G1 cell cycle synchronization on improving KI efficiency 43
2.4.3. Low concentration HU selection for progressive shift of cell cycle and improving KI efficiency 44
2.4.4. Enhancement of SSI efficiency using modified donor plasmid 50
2.4.5. Establishment of double KI monitoring cell lines 53
2.4.6. Simultaneous and sequential dual-loci KI using DCD system 56
2.4.7. Effect of different CRISPR-HDR vector configuration on KI efficiency in CHO Cells 58
2.4.8. Controlling the relative ratios of CRISPR-HDR component using DCD and MC system 60
2.4.9. Application of DCD and MC system for simultaneous KI 63
2.4.10. Generation of engineered CHO cell line using simultaneous KI of Tet-On3G inducible elements 65
2.5. Discussion 67
CHAPTER 3. Development of a sustainable and quantitative transgene expression platform for reliable mammalian cell engineering with gain-of-function mutations 69
3.1. Abstract 70
3.2. Introduction 71
3.3. Materials and Methods 74
3.3.1. Molecular cloning and plasmid construction 74
3.3.2. Cell culture and maintenance 101
3.3.3. Cell transfection 101
3.3.4. Single Nucleotide polymorphism (SNP) correction 102
3.3.5. Generation of SSI-based Tet-On3G-inducible MCLs 103
3.3.6. Generation of selection-based ROSE RI pools 104
3.3.7. Doxycycline induction for stability test and expression dynamics 105
3.3.8. RMCE of effector genes 105
3.3.9. Application of the ROSE platform for GOF mutation with tunable expression and timing 106
3.3.10. Preparation of gDNA, RNA, and cDNA 108
3.3.11. PCR for clonal validation and qRT-PCR for relative gene copy number and mRNA expression analysis 108
3.3.12. Bisulfite conversion sequencing for methylation state 109
3.3.13. Cold capture assay for the surface staining of proteins 109
3.3.14. Fluorescence expression analysis using imaging and flow cytometry 110
3.3.15. Western blotting analysis and measurement of antibody concentrations via ELISA 110
3.3.16. Statistical analyses 113
3.4. Results 114
3.4.1. SSI-based Tet-On inducible MCLs exhibit reproducible but unsustainable induction capacity 114
3.4.2. Progressive genetic circuit malfunction in CMV-Tet-On3G-inducible MCLs is associated with the promoter methylation of Tet-On3G 120
3.4.3. Decline in the relative Tet-On3G transcript levels indicates the inherent susceptibility of inducible CLD to genetic circuit malfunction 126
3.4.4. Modular design of the vector constructs and integration sites significantly influences the induction performance and robustness of CLD strategies 131
3.4.5. ROSE MCLs exhibit sophisticated and dynamic transgene expression control 139
3.4.6. Quantitative and sustainable regulation of effector genes in ROSE MCLs via RMCE results in precise GOF mutations 144
3.4.7. RMCE of transgene encoding therapeutic antibody in ROSE MCLs leads to controlled recombinant antibody production 151
3.4.8. ROSE platform-dependent expression control enables difficult-to-express bsAb production via biphasic culture 157
3.4.9. ROSE platform-dependent expression control of the transcription factor, Blimp1, aids in biomanufacturing 163
3.4.10. Engineering effect of DDR-related gene overexpression on CRISPR/Cas9-mediated SSI efficiency 172
3.5. Discussion 176
CHAPTER 4. RMCE-based Genome-wide CRISPR screening for the development of advanced CHO factory 179
4.1. Abstract 180
4.2. Introduction 181
4.3. Materials and Methods 186
4.3.1. Molecular cloning 186
4.3.2. CHO genome-wide CRISPR KO gRNA library construction 186
4.3.3. Cell lines, culture maintenance, and culture media 191
4.3.4. Generation of the T2 site LP MCLs 192
4.3.5. Generation of cell-based gRNA libraries 194
4.3.6. Generation of cell-based KO libraries 198
4.3.7. HP cell population enrichment and target validation 201
4.3.8. Preparation of NGS sample 205
4.3.9. NGS data analysis 205
4.4. Results and discussion 207
4.4.1. Amplicon sequencing read quality validation and assessment of gRNA library representation 207
4.4.2. Identification and characterization of essential genes in host and recombinant CHO cells 211
4.4.3. Identification of novel targets using FACS-based CRISPR screen 215
CONCLUSION 217
REFERENCES 219
ABSTRACT IN KOREAN 235
