Artificial Integration Sites Harboring Ubiquitous Chromatin Opening Elements Enable Efficient Targeted Knock-In and High Transgene Expression in Mammalian Cells
- 주제(키워드) Chinese hamster ovary (CHO) , CRISPR/Cas9 , Hot spot , Knock-in , Ubiquitous Chromatin Opening Element
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 이재성
- 발행년도 2022
- 학위수여년월 2022. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000031971
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
CRISPR/Cas9-mediated targeted gene integration (TI) has been used to generate recombinant mammalian cell lines with predictable transgene expression. Identifying genomic hot spots that render high and stable transgene expression and knock-in (KI) efficiency is critical for fully implementing TI-mediated cell line development; however, such identification is cumbersome. In this study, we developed an artificial KI construct that can be used as a hot spot at different genomic loci. The ubiquitous chromatin opening element (UCOE) was employed because of its ability to open chromatin and enable stable and site-independent transgene expression. UCOE KI cassettes were randomly integrated into CHO-K1 and HEK293T cells, followed by TI of EGFP onto the artificial UCOE KI site. The CHO-K1 random pool harboring 5’2.2A2UCOE-CMV displayed a significant increase in EGFP expression level and KI efficiency, and a markedly lower CV than that of the control without UCOE. In addition, 5’2.2A2UCOE-CMV showed improved Cas9 accessibility in the HEK293T genome, leading to an increase in indel frequency and homology-independent KI. Integration of 5’2.2A2UCOE-CMV into the repressed locus induced comparable EGFP expression levels and KI efficiency to the known human safe harbor site, despite being KI method-dependent and not significantly different from CMV. Overall, this assessment revealed the potential of UCOE KI constructs as artificial integration sites in streamlining the screening of high-production targeted integrants by mitigating the selection of genomic hot spots.
more목차
1. Introduction 1
2. Materials and Methods 5
2.1. Plasmid construction 5
2.2. Cell lines and culture maintenance 7
2.3. Generation of artificial KI construct random pools 7
2.4. Generation and validation of artificial KI construct clones 8
2.5. Tracking of Indels by DEcomposition (TIDE) analysis 9
2.6. CRISPR/Cas9-mediated KI 9
2.7. Recombinase-mediated KI 10
2.8. Long-term culture for assessing the stability of recombinant cells 10
2.9. Flow cytometry 10
2.10. Statistical analysis 11
3. Results 21
3.1. Construction of artificial KI constructs harboring A2UCOE 21
3.2. 5’2.2A2UCOE-CMV randomly integrated sites are the most active and genome editing-prone in CHO-K1 and HEK293T cells 25
3.3. 5’2.2A2UCOE-CMV randomly integrated sites enable stable transgene expression and enrichment of high-producing populations 31
3.4. Estimation of the targeted KI efficiency at artificial KI constructs harboring A2UCOE at the defined locus 34
4. Discussion 42
5. References 46
