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Reprogramming amino acid catabolism in CHO cells with CRISPR-Cas9 genomeediting improves cell growth and reduces by-product secretion

Ley, Daniel; Pereira, Sara; Pedersen, Lasse Ebdrup; Arnsdorf, Johnny; Hefzi, Hooman; Lund, AnneMathilde; Kwang Ha, Tae; Wulff, Tune; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

Publication date:2017

Link back to DTU Orbit

Citation (APA):Ley, D., Pereira, S., Pedersen, L. E., Arnsdorf, J., Hefzi, H., Lund, A. M., Kwang Ha, T., Wulff, T., Kildegaard, H.F., & Andersen, M. R. (2017). Reprogramming amino acid catabolism in CHO cells with CRISPR-Cas9 genomeediting improves cell growth and reduces by-product secretion.

Daniel Ley1,2, Sara Pereira2, Lasse Ebdrup Pedersen2, Johnny Arnsdorf2, Hooman Hefzi3,4, Anne Mathilde Lund1, Tae Kwang Ha2, Tune Wul�2, Helene Faustrup Kildegaard2, Mikael Rørdam Andersen1.

(1) Network Engineering of Eukaryotic Cell Factories, Department of Bioengineering and Biomedicine, Technical University of Denmark, Kgs. Lyngby, Denmark; (2) Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark; (3) Department of Bioengineering, University of California, San Diego, United States; (4) Novo Nordisk Foundation Center for Biosustainability at the University of California, San Diego, School of Medicine, United States.Correspondence: Daley@biosustain.dtu.dk / Mr@bio.dtu.dk

Reprogramming Amino Acid Catabolism in CHO Cells with CRISPR-Cas9 Genome Editing Improves Cell Growth and

Reduces By-Product Secretion

Figure here!

References1. Mulukutla, B. C. et al. (2017), Biotechnology and Bioengineering, 114(8), pp. 1779-1790.2. Ahn, W.S. & Antoniewicz, M. R. (2012), Biotechnology Journal, 7, pp. 61-74.3. Templeton N. et al. (2013), Biotechnology and Bioengineering, 110(7), pp. 2013-2024.4. Ley & Kazemi et al. (2015), Biotechnology and Bioengineering,112(11), pp. 2373-2387.5. Kildegaard et al. (2013), Current opinion in biotechnology, 24, pp. 1102-1107.

CHO cells primarily utilize amino acids for three processes: biomass synthesis, recombinant protein production and catabolism. In this work, we disrupted 9 amino acid catabolic genes participating in 7 di�erent catabolic pathways, to increase synthesis of biomass and recombinant protein, while reducing production of growth-inhibiting metabolic by-products from amino acid catabolism.

Key message

Overview of experimentsBackground

Conclusion

Physiology of single gene disrupted CHO cells

Disruption of single amino acid catabolic pathways in CHO cells reduces speci�c production of lactate and ammonium, while increasing µmax and IVCD, leading to increased titers of recombi-nant proteins. Disruption of multiple catabolic pathways further reduces secretion of lactate and ammonium, but does not increase growth. Thus, we recommend combinatorial disruption of multiple amino acid catabolic pathways, to identify a set of disruptions that increase growth, while reducing secretion of lactate and ammonium.

To study the physiological impact of disrupting single amino acid catabolic pathways, we char-acterized single gene disrupted clones in triplicate shake �ask cultures in batch mode. We moni-tored physiological changes in terms of maximum speci�c growth rate (µmax), integral of viable cell density (IVCD) and secretion of lactate and ammonium.

To exclude that the improved phenotypes are caused by clonal variation, we characterized mul-tiple clones with di�erent mutations in gene 4 and 6, and found a strong link between genotype and phenotype.

To investigate the impact of EPO production on metabolism, we reconstructed glycolysis, TCA and amino acid catabolism in CHO cells (see link below) and integrated di�erential gene expression data from phase II of chemostat culture. The data integration revealed decreased transcription level of genes responsible for degradation of the amino acids most frequently found in EPO. Thus, indicating possible regulatory adaptation of gene expression towards decreased amino acid catabolism, spe-ci�c for the most abundant amino acids in EPO, in the high producer relative to the low producer.

AcknowledgementsWe acknowledge Karen Katrine Brøndum and Zu�ya Suk-hova for technical assistance with generation of genome edited cell lines. Moreover, we thank Sara Bjørn Petersen for cloning plasmids and Thomas Beuchert Kallehauge for sharing his experience in design of quantitative PCR experi-ments and Lene Holberg Blicher for assisting in the pro-teomics experiment. The Novo Nordisk Foundation provid-ed funding for this work.

Amino acid catabolism produces a wide range of growth inhibiting compounds1, amongst these ammonium and lactate. Ammonium is produced by transamination and deamination re-actions2, whereas lactate is produced by either amino acid catabolic pathways fueling glycolysis or by NADH producing catabolic pathways, which forces the cell to regenerate NAD+ through lactate synthesis3. Disruption of amino acid catabolic pathways may reduce production of growth-inhibiting metabolic by-products.

Validation of functional gene knock-out

Physiology of multiple gene disrupted CHO cells

Target genes were identi�ed using a metabolic network reconstruction of amino acid catabo-lism4. Gene knock-out was performed with CRISPR-Cas9. Single cells expressing GFP-linked Cas9 were enriched on FACS. Physiology of gene-edited clones was assessed in shake flasks and bioreactors. Phenotypes were validated by targeted genome sequencing, qRT-PCR, western blot and proteomic analysis.

TCA cycleAmino acid transamination Glutamate

NAD+NADH

NH4+

Pyruvate Lactate

GlycolysisAmino acid

NAD+ NADH

NAD+ NADH

Amino acid

NAD+NADHNAD+NADH NH4+

catabolic pathway

Functional gene disruptions were validated using deep sequencing of the targeted genomic loci, gene expression analysis, western blots and proteomics. All genes displayed out-of-frame mutations (A) and generally reduced transcription (B). Western blots indicated potential wild type proteins in some clones (C), so proteomic analysis and mRNA sequencing was applied to verify functional knock-out of target genes (ongoing work).

IgG vs WT

sgRNA

GFP-2A-Cas9

Data integration Transfection Single cell sorting Expansion and characterizationTarget selection

• Growth performance• Metabolite pro�les

Clone validation• Genomics• Transcript analyses• Protein analyses

1 2 3 4 5 6 7 8 950

100

150

Nor

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ized

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pe(W

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Maximum specific growth rate

* * **

1 2 3 4 5 6 7 8 950

100

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* *

1 2 3 4 5 6 7 8 950

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*

Single gene disrupted clones generally showed an increased growth phenotype with 8 of 9 clones displaying increased µmax (up to 115% of WT), while 6 of 9 clones had increased IVCD (up to 136% of WT). Speci�c secretion of lactate was reduced in 4 of 9 clones (down to 81% of WT), while speci�c secretion of ammonium was reduced in 5 of 9 clones (down to 91% of WT). Mono-clonal antibody titers increased proportionally to IVCD (data not shown).

Target gene: 1 2 3 4 5 6 7 8 9 Indel sizes: -1 +1 -29/-16 +1 +28 -13 +1 -22 -20

A

B C

Gene1

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Gene1

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Gene7

- 3'en

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Gene8

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Gene8

- 3'en

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Gene9

- 5'en

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Gene9

- 3'en

d0.0

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xpre

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2.0

To explore potential synergistic e�ects of disrupting multiple pathways, we targeted gene 1-4 for knock-out, but did not achieve full knock-out of all genes. Still, we isolated two clones with interesting genotypes. Clones were characterized in duplicated bioreactor cultures and showed further reduced lactate and ammonium secretion, but no growth bene�t.

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Growth - Gene 4 Growth - Gene 6

4

Target gene: 1 2 3 4 Indel sizes: -1/-10 -9/WT +98/+105 +1

Clone 1Target gene: 1 2 3 4 Indel sizes: -1/-10 WT +98/+105 +1

Clone 2

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[pm

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30 % decrease

14 % decrease

Specific ammonium secretion rate

Clone 1

Clone 2 WT0

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Lact

ate

secr

etio

n ra

te [p

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] 22 % decrease

22 % decrease

Specific lactate secretion rate

Figure graphics were modi�ed from5..

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Gene 1

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WT Gene 9

Growth and viability