CRISPR/Cas12a-mediated CHO genome engineering can be effectively integrated at multiple stages of the cell line generation process for bioproduction.

Advanced Search

Patrick G Schweickert, Ning Wang, Stephanie L Sandefur, Michael E Lloyd, Stephen F Konieczny, Christopher C Frye, Zhuo Cheng

Author Information

Patrick G Schweickert: Department of Biological Sciences and the Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana, USA.
Ning Wang: Bioprocess Research and Development, Eli Lilly and Company, Indianapolis, Indiana, USA.
Stephanie L Sandefur: Bioprocess Research and Development, Eli Lilly and Company, Indianapolis, Indiana, USA.
Michael E Lloyd: Bioprocess Research and Development, Eli Lilly and Company, Indianapolis, Indiana, USA.
Stephen F Konieczny: Department of Biological Sciences and the Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana, USA.
Christopher C Frye: Bioprocess Research and Development, Eli Lilly and Company, Indianapolis, Indiana, USA.
Zhuo Cheng: Bioprocess Research and Development, Eli Lilly and Company, Indianapolis, Indiana, USA. ORCID

PMID: 33369118 DOI: 10.1002/biot.202000308

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.

CHO CRISPR Cas12a Chinese hamster ovary Cpf1

Hacker, D. L., De Jesus, M., & Wurm, F. M. (2009). 25 Years of recombinant proteins from reactor-grown cells - Where do we go from here? Biotechnol. Adv., 27, 1023-1027. https://doi.org/10.1016/j.biotechadv.2009.05.008

Schweickert, P. G., & Cheng, Z. (2019). Application of genetic engineering in biotherapeutics development. J. Pharm. Innov., 15, 232-254. https://doi.org/10.1007/s12247-019-09411-6

Lewis, N. E., Liu, X., Li, Y., Nagarajan, H., Yerganian, G., O'brien, E., … Palsson, B. O. (2013). Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome. Nat. Biotechnol., 31, 759-765. https://doi.org/10.1038/nbt.2624

Xu, X., Nagarajan, H., Lewis, N. E., Pan, S., Cai, Z., Liu, X., … Wang, J. (2011). The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat. Biotechnol., 29, 735-741. https://doi.org/10.1038/nbt.1932

Adli, M. (2018). The CRISPR tool kit for genome editing and beyond. Nat. Commun., 9, 1-13. https://doi.org/10.1038/s41467-018-04252-2

Carroll, D. (2011). Genome engineering with zinc-finger nucleases. Genetics, 188, 773-782. https://doi.org/10.1534/genetics.111.131433

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptice bacterial immunity. Science, 337, 816-821. https://doi.org/10.1126/science.1225829

Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., … Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339, 819-823. https://doi.org/10.1126/science.1231143.Multiplex

Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., … Zhang, F. (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163, 759-771. https://doi.org/10.1016/j.cell.2015.09.038

Liu, J.-J., Orlova, N., Oakes, B. L., Ma, E., Spinner, H. B., Baney, K. L. M., … Doudna, J. A. (2019). CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature, 566, 218-223. https://doi.org/10.1038/s41586-019-0908-x

Swarts, D. C., & Jinek, M. (2018). Cas9 versus Cas12a/Cpf1: Structure - function comparisons and implications for genome editing. WIREs RNA, 9, 1-19. https://doi.org/10.1002/wrna.1481

Swarts, D. C., & Jinek, M. (2019). Mechanistic insights into the cis- and trans-acting DNase activities of Cas12a. Mol. Cell., 73(3), 589-600.e4. https://doi.org/10.1016/j.molcel.2018.11.021

Schmieder, V., Bydlinski, N., Strasser, R., Baumann, M., Kildegaard, H. F., Jadhav, V., & Borth, N. (2018). Enhanced genome editing tools for multi-gene deletion knock-out approaches using paired CRISPR sgRNAs in CHO cells. Biotechnol. J., 13, 1-10. https://doi.org/10.1002/biot.201700211

Kim, D., Kim, J., Hur, J. K., Been, K. W., Yoon, S.-H., & Kim, J.-S. (2016). Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat. Biotechnol., 34, 863-868. https://doi.org/10.1038/nbt.3609

Strohkendl, I., Saifuddin, F. A., Rybarski, J. R., Finkelstein, I. J., & Russell, R. (2018). Kinetic basis for DNA target specificity of CRISPR-Cas12a. Mol. Cell, 71, 816-824.e3. https://doi.org/10.1016/j.molcel.2018.06.043

Swarts, D. C., Van Der Oost, J., & Jinek, M. (2017). Structural basis for guide RNA processing and seed-dependent DNA targeting by CRISPR-Cas12a. Mol. Cell, 66, 221-233.e4. https://doi.org/10.1016/j.molcel.2017.03.016

Chenouard, V., Brusselle, L., Heslan, J.-M., Remy, S., Ménoret, S., Usal, C., … Tesson, L. (2016). A rapid and cost-effective method for genotyping genome-edited animals: A heteroduplex mobility assay using microfluidic capillary electrophoresis. J. Genet. Genomics, 43, 341-348. https://doi.org/10.1016/j.jgg.2016.04.005

Lonowski, L. A., Narimatsu, Y., Riaz, A., Delay, C. E., Yang, Z., Niola, F., … Frödin, M. (2017). Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis. Nat. Protoc., 12, 581-603. https://doi.org/10.1038/nprot.2016.165

Krebs, L., Gao, J., & Frye, C. (2015). Statistical verification that one round of fluorescence-activated cell sorting (FACS) can effectively generate a clonally-derived cell line. Bioprocess. J., 13, 6-19. https://doi.org/10.12665/j134.krebs

Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., … Zhang, F. (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol., 31, 827-832. https://doi.org/10.1038/nbt.2647

Haeussler, M., Schönig, K., Eckert, H., Eschstruth, A., Mianné, J., Renaud, J.-B., … Concordet, J.-P. (2016). Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol., 17, 148. https://doi.org/10.1186/s13059-016-1012-2

Cost, G. J., Freyvert, Y., Vafiadis, A., Santiago, Y., Miller, J. C., Rebar, E., … Gregory, P. D. (2010). BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cells. Biotechnol. Bioeng., 105, 330-340. https://doi.org/10.1002/bit.22541

Bin Moon, S., Lee, J. M., Kang, J. G., Lee, N.-E., Ha, D.-I., Kim, D. Y., … Kim, Y.-S. (2018). Highly efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a uridinylate-rich 3′-overhang. Nat. Commun., 9, 3651. https://doi.org/10.1038/s41467-018-06129-w

Gutiérrez, A. H., Moise, L., & De Groot, A. S. (2012). Of [hamsters] and men: A new perspective on host cell proteins. Hum. Vaccines Immunother., 8, 1172-1174. https://doi.org/10.4161/hv.22378

Hall, T., Sandefur, S. L., Frye, C. C., Tuley, T. L., & Huang, L. (2016). Polysorbates 20 and 80 degradation by group XV lysosomal phospholipase A2 isomer X1 in monoclonal antibody formulations. J. Pharm. Sci., 105, 1633-1642. https://doi.org/10.1016/j.xphs.2016.02.022

Wang, X., Hunter, A. K., & Mozier, N. M. (2009). Host cell proteins in biologics development: Identification, quantitation and risk assessment. Biotechnol. Bioeng., 103, 446-458. https://doi.org/10.1002/bit.22304

Valente, K. N., Levy, N. E., Lee, K. H., & Lenhoff, A. M. (2018). Applications of proteomic methods for CHO host cell protein characterization in biopharmaceutical manufacturing. Curr. Opin. Biotechnol., 53, 144-150. https://doi.org/10.1016/j.copbio.2018.01.004

Zhang, Q., Goetze, A. M., Cui, H., Wylie, J., Trimble, S., Hewig, A., & Flynn, G. C. (2014). Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry. MAbs, 6, 659-670. https://doi.org/10.4161/mabs.28120

Laux, H., Romand, S., Nuciforo, S., Farady, C. J., Tapparel, J., Buechmann-Moeller, S., … Bodendorf, U. (2018). Degradation of recombinant proteins by Chinese hamster ovary host cell proteases is prevented by matriptase-1 knockout. Biotechnol. Bioeng., 115, 2530-2540. https://doi.org/10.1002/bit.26731

Chiu, J., Valente, K. N., Levy, N. E., Min, L., Lenhoff, A. M., & Lee, K. H. (2017). Knockout of a difficult-to-remove CHO host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations. Biotechnol. Bioeng., 114, 1006-1015. https://doi.org/10.1002/bit.26237

Yamane-Ohnuki, N., Kinoshita, S., Inoue-Urakubo, M., Kusunoki, M., Iida, S., Nakano, R., … Satoh, M. (2004). Establishment of FUT8 knockout Chinese hamster ovary cells: An ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol. Bioeng., 87, 614-622. https://doi.org/10.1002/bit.20151

Animals

CHO Cells

CRISPR-Cas Systems

Clustered Regularly Interspaced Short Palindromic Repeats

Cricetinae

Cricetulus

Gene Editing

Journal Article

OpenLB
Open Library of Bioscience