CRISPR/Cas9-Mediated Knockout of MicroRNA-744 Improves Antibody Titer of CHO Production Cell Lines

Biotechnology Journal
Nadja RaabKerstin Otte

Abstract

MicroRNAs (miRNAs) are noncoding RNAs that serve as versatile molecular engineering tools to improve production cells by overexpression or knockdown of miRNAs showing beneficial or adverse effects on cell-culture performance. The genomic knockout (KO) of noncoding RNAs in Chinese hamster ovary (CHO) production cells has not been reported. However, given the significant number of miRNAs showing negative effects on CHO-bioprocess performance and the development of clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas9), genome editing tools facilitate precise optimization of CHO cells via modulation of noncoding RNAs. In a previous high-content miRNA screen, miR-744 was identified as a potential target associated with reduced productivity. Hence, the genomic miR-744 precursor sequence is deleted by two single guide RNA (sgRNA)-Cas9-mediated DNA double-strand breaks (DSB) flanking the miR-744 locus. After fluorescence-activated cell sorting (FACS), clonal miR-744 KO cell lines are recovered and three of them are confirmed as miR-744 KOs. Impacts of CRISPR/Cas9 editing are characterized at the genetic, transcript, and phenotypic levels. During batch cultivation, antibody titers of miR-744 ...Continue Reading

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Citations

Jul 12, 2019·Applied Microbiology and Biotechnology·Changzhi XuBuchang Zhang
Apr 30, 2020·Biotechnology and Bioengineering·Ioanna TzaniColin Clarke
Jan 11, 2020·Frontiers in Bioengineering and Biotechnology·Nagesh K Tripathi, Ambuj Shrivastava
Dec 11, 2020·Biotechnology Progress·Ann-Cathrin LerouxChristoph Zehe
Mar 12, 2021·Biochemical Society Transactions·Roberto DoniniCleo Kontoravdi

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