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Implementation of ubiquitous chromatin opening elements as artificial integration sites for CRISPR/Cas9-mediated knock-in in mammalian cellsoa mark
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Publication Year
2023-04-01
Journal
Engineering in Life Sciences
Publisher
John Wiley and Sons Inc
Citation
Engineering in Life Sciences, Vol.23 No.4
Keyword
Chinese hamster ovary (CHO)CRISPR/Cas9hot spotknock-inubiquitous chromatin opening element
Mesh Keyword
Chinese hamster ovaryCRISPR/cas9Enhanced green fluorescent proteinGenomicsHotspotsKnock-inMammalian cellsTransgene expressionUbiquitous chromatin opening element
All Science Classification Codes (ASJC)
BiotechnologyBioengineeringEnvironmental Engineering
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 (CLD); 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 enhanced green fluorescent protein (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 compared with 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. 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.
ISSN
1618-2863
Language
eng
URI
https://aurora.ajou.ac.kr/handle/2018.oak/33297
https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85150518505&origin=inward
DOI
https://doi.org/10.1002/elsc.202200047
Journal URL
http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1618-2863
Type
Article
Funding
This work was supported by the NRF funded by the Korean government (2021R1A2C4002733 and 2019R1A6A1A11051471). The authors declare no competing financial interests.
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Lee, Jae Seong이재성
College of Bio-convergence Engineering
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