CRISPR/Cas9‐Mediated Knockout of MicroRNA‐744 Improves Antibody Titer of CHO Production Cell Lines. Issue 5 (15th April 2019)

• Record Type: Journal Article
• Authors: Raab, Nadja; Mathias, Sven; Alt, Kerstin; Handrick, René; Fischer, Simon; Schmieder, Valerie; Jadhav, Vaibhav; Borth, Nicole; Otte, Kerstin
• Is Part Of: Biotechnology journal. Volume 14:Issue 5(2019)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2019-04-15
• Subjects: cell line engineering -- CHO -- CRISPR/Cas9 -- knockout -- microRNAs; Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.201800477
• Languages: English
• ISSNs: 1860-6768

Abstract

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 KOs are significantly increased to 190–311 mg L −1 compared to a nontargeting (NT) sgRNA transfected clonal control with 156 mg L −1, pointing towards the potential of miRNA KO for cell line engineering. Abstract : The current study applied a novel strategy to increase product yields of Chinese hamster ovary (CHO) cells using clustered regularly interspaced short palindromic repeats/CRISPR‐associated proteins (CRISPR/Cas9)–mediated deletion of miR‐744, a microRNA having adverse effects on productivity. Obtained miR‐744 knockout (KO) clones are characterized on genetic, transcript, and phenotypic level to prove functionality of miR‐744 KO and effects on productivity.