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Artificial Integration Sites Harboring Ubiquitous Chromatin Opening Elements Enable Efficient Targeted Knock-In and High Transgene Expression in Mammalian Cells
- Author(s)
- 김슬미
- Advisor
- 이재성
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2022-08
- Language
- eng
- Keyword
- CRISPR/Cas9; Chinese hamster ovary (CHO); Hot spot; Knock-in; Ubiquitous Chromatin Opening Element
- Alternative Abstract
- 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.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)
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