期刊
BIOTECHNOLOGY JOURNAL
卷 16, 期 4, 页码 -出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.202000308
关键词
Cas12a; Chinese hamster ovary; CHO; Cpf1; CRISPR
The study demonstrates the successful application of CRISPR/Cas12a for generating CHO KO cell lines with improved product quality, with efficiency dependent on the targeting RNA used; small screens of clonally derived cell lines can be used to generate CHO KO cell lines, and a novel bulk culture analysis approach is proposed for assessing CRISPR RNA efficiency and determining ideal screen sizes; Cas12a can be directly integrated into the cell line generation process with no negative impact on titer or screen size.
Most biopharmaceuticals produced today are generated using Chinese hamster ovary (CHO) cells, therefore significant attention is focused on methods to improve CHO cell productivity and product quality. The discovery of gene-editing tools, such as CRISPR/Cas9, offers new opportunities to improve CHO cell bioproduction through cell line engineering. Recently an additional CRISPR-associated protein, Cas12a (Cpf1), was shown to be effective for gene editing in eukaryotic cells, including CHO. In this study, we demonstrate the successful application of CRISPR/Cas12a for the generation of clonally derived CHO knockout (KO) cell lines with improved product quality attributes. While we found Cas12a efficiency to be highly dependent on the targeting RNA used, we were able to generate CHO KO cell lines using small screens of only 96-320 clonally derived cell lines. Additionally, we present a novel bulk culture analysis approach that can be used to quickly assess CRISPR RNA efficiency and determine ideal screen sizes for generating genetic KO cell lines. Most critically, we find that Cas12a can be directly integrated into the cell line generation process through cotransfection with no negative impact on titer or screen size. Overall, our results show CRISPR/Cas12a to be an efficient and effective CHO genome editing tool.
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