Efficient CRISPR/Cas9-mediated multiplex genome editing in CHO cells via high-level sgRNA-Cas9 complex

Abstract

Increasing demand for recombinant therapeutic proteins has warranted the need for an efficient host cell to produce high-quality proteins, with a high yield. Chinese hamster ovary (CHO) cells appear to meet this demand, and their genetic tailoring will facilitate improvements in their productivity for recombinant proteins. Recent advances in programmable RNA-guided Cas9 nuclease (RGN) have facilitated CHO cell engineering via site-specific genome editing. One critical determinant for increasing genomeediting efficiency is attaining a balanced expression level of Cas9 nuclease and guide RNAs in the nucleus. Here, we achieved high-level expression of Cas9 nuclease and single guide RNA (sgRNA), enhancing expression levels approximately three-fold over the conventional methodology by using an iterative transfection approach. We demonstrated that high abundance of sgRNA and Cas9 nuclease induced a two-fold increase in the site-specific mutation rate on average for both single and multiple genetic targets. Sequencing results confirmed frame-shift mutations at targeted genomic loci created by error-prone NHEJassociated mutations. Moreover, we controlled the amount of sgRNA-Cas9 complex formation in vitro and delivered the complex directly to cells, resulting in the maximization of mutation frequency by the high-level of sgRNA-Cas9 complex. Importantly, mutation rates of putative off-target sites remained minimal in spite of the improved genome-editing efficiency. These results provide an efficient strategy for editing the CHO genome with the reduction of the time-consuming screening efforts aimed at isolating clones with desirable properties.

This is a preview of subscription content, log in to check access.

References

  1. 1.

    Datta, P., R. J. Linhardt, S. T. Sharfstein (2013) An ’omics approach towards CHO cell engineering. Biotechnol. Bioeng. 110: 1255–1271.

    CAS  Article  Google Scholar 

  2. 2.

    Steentoft, C., Vakhrushev, S. Y., Vester-Christensen, M. B., Schjoldager, K. T., Kong, Y., Bennett, E. P., U. Mandel, H. Wandall, S. B. Levery, and H. Clausen (2011) Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines. Nat. Methods. 8: 977–982.

    CAS  Article  Google Scholar 

  3. 3.

    Meuris, L., F. Santens, G. Elson, N. Festjens, M. Boone, A. Dos Santos, S. Devos, F. Rousseau, E. Plets, E. Houthuys, P. Malinge, G. Magistrelli, L. Cons, L. Chatel, B. Devreese, and N. Callewaert (2014) GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins. Nat. Biotechnol. 32: 485–489.

    CAS  Article  Google Scholar 

  4. 4.

    Ronda, C., L. E. Pedersen, H. G. Hansen, T. B. Kallehauge, M. J. Betenbaugh, A. T. Nielsen, and H. F. Kildegaard (2014) Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool. Biotechnol. Bioeng. 111: 1604–1616.

    CAS  Article  Google Scholar 

  5. 5.

    Xu, X., H. Nagarajan, N. E. Lewis, S. Pan, Z. Cai, X. Liu, W. Chen, M. Xie, W. Wang, S. Hammond, M.R. Andersen, N. Neff, B. Passarelli, W. Koh, H. C. Fan, J. Wang, Y. Gui, K. H. Lee, M. J. Betenbaugh, S. R. Quake, I. Famili, B. O. Palsson, and J. Wang (2011) The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat. Biotechnol. 29: 735–741.

    CAS  Article  Google Scholar 

  6. 6.

    Lewis, N. E., X. Liu, Y. Li, H. Nagarajan, G. Yerganian, E. O’Brien, A. Bordbar, A. M. Roth, J. Rosenbloom, C. Bian, M. Xie, W. Chen, N. Li, D. Baycin-Hizal, H. Latif, J. Forster, M. J. Betenbaugh, I. Famili, X. Xu, J. Wang, and B. O. Palsson (2013) Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome. Nat. Biotechnol. 31: 759–765.

    CAS  Article  Google Scholar 

  7. 7.

    Sealover, N. R., A. M. Davis, J. K. Brooks, H. J. George, K. J. Kayser, and N. Lin (2013) Engineering Chinese hamster ovary (CHO) cells for producing recombinant proteins with simple glycoforms by zinc-finger nuclease (ZFN)-mediated gene knockout of mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (Mgat1). J. Biotechnol. 167: 24–32.

    CAS  Article  Google Scholar 

  8. 8.

    Carlson, D. F., S. C. Fahrenkrug, and P. B. Hackett (2012) Targeting DNA With Fingers and TALENs. Mol. Ther. Nucleic acids. 1: e3.

  9. 9.

    Sander, J. D. and J. K. Joung (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32: 347–355.

    CAS  Article  Google Scholar 

  10. 10.

    Mali, P., L. Yang, K. M. Esvelt, J. Aach, M. Guell, J. E. DiCarlo, J. E. Norville, and G. M. Church (2013) RNA-guided human genome engineering via Cas9. Science. 339: 823–826.

    CAS  Article  Google Scholar 

  11. 11.

    Cong, L., F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. A. Marraffini, and F. Zhang (2013) Multiplex genome engineering using CRISPR/Cas systems. Sci. 339: 819–823.

    CAS  Article  Google Scholar 

  12. 12.

    Deveau, H., J. E. Garneau, and S. Moineau (2010) CRISPR/Cas system and its role in phage-bacteria interactions. Ann. Rev. Microbiol. 64: 475–493.

    CAS  Article  Google Scholar 

  13. 13.

    Cho, S. W., S. Kim, J. M. Kim, and J. S. Kim (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31: 230–232.

    CAS  Article  Google Scholar 

  14. 14.

    Fu, Y., J. A. Foden, C. Khayter, M. L. Maeder, D. Reyon, J. K. Joung, and J. D. Sander (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31: 822–826.

    CAS  Article  Google Scholar 

  15. 15.

    Jiang, W., D. Bikard, D. Cox, F. Zhang, and L. A. Marraffini (2013) RNA-guided editing of bacterial genomes using CRISPRCas systems. Nat. Biotechnol. 31: 233–239.

    CAS  Article  Google Scholar 

  16. 16.

    Hsu, P. D., E. S. Lander, and F. Zhang (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell. 157: 1262–1278.

    CAS  Article  Google Scholar 

  17. 17.

    Ramakrishna, S., A. B. Kwaku Dad, J. Beloor, R. Gopalappa, S. K. Lee, and H. Kim (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 24: 1020–1027.

    CAS  Article  Google Scholar 

  18. 18.

    Duda, K., L. A. Lonowski, M. Kofoed-Nielsen, A. Ibarra, C. M. Delay, Q. Kang, Z. Yang, S. M. Pruett-Miller, E. P. Bennett, H. H. Wandall, G. D. Davis, S. H. Hansen, and M. Frodin (2014) Highefficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs. Nucleic Acids Res. 42: e84.

    CAS  Article  Google Scholar 

  19. 19.

    Kim, N. S., T. H. Byun, and G. M. Lee (2001) Key determinants in the occurrence of clonal variation in humanized antibody expression of cho cells during dihydrofolate reductase mediated gene amplification. Biotechnol. Progr. 17: 69–75.

    Article  Google Scholar 

  20. 20.

    Ghorbaniaghdam, A., J. Chen, O. Henry, and M. Jolicoeur (2014) Analyzing clonal variation of monoclonal antibody-producing CHO cell lines using an in silico metabolomic platform. PloS One. 9: e90832.

  21. 21.

    Shalem, O., N. E. Sanjana, E. Hartenian, X. Shi, D. A. Scott, T. S. Mikkelsen, D. Heckl, B. L. Ebert, D. E. Root, J. G. Doench, and F. Zhang (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Sci. 343: 84–87.

    CAS  Article  Google Scholar 

  22. 22.

    Santiago, Y., E. Chan, P. Q. Liu, S. Orlando, L. Zhang, F. D. Urnov, M. C. Holmes, D. Guschin, A. Waite, J. C. Miller, E. J. Rebar, P. D. Gregory, A. Klug, and T. N. Collingwood (2008) Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 105: 5809–5814.

    CAS  Article  Google Scholar 

  23. 23.

    McLaughlin, P. J., B. Bakall, J. Choi, Z. Liu, T. Sasaki, E. C. Davis, A. D. Marmorstein, and L. Y. Marmorstein (2007) Lack of fibulin-3 causes early aging and herniation, but not macular degeneration in mice. Hum. Mol. Genet. 16: 3059–3070.

    CAS  Article  Google Scholar 

  24. 24.

    Ranjan, V., R. Waterbury, Z. Xiao, and S. L. Diamond (1996) Fluid shear stress induction of the transcriptional activator c-fos in human and bovine endothelial cells, HeLa, and Chinese hamster ovary cells. Biotechnol. Bioeng. 49: 383–390.

    CAS  Article  Google Scholar 

  25. 25.

    Zuris, J. A., D. B. Thompson, Y. Shu, J. P. Guilinger, J. L. Bessen, J. H. Hu, M. L. Maeder, J. K. Joung, Z. Y. Chen, and D. R. Liu (2015) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33: 73–80.

    CAS  Article  Google Scholar 

  26. 26.

    Pattanayak, V., S. Lin, J. P. Guilinger, E. Ma, J. A. Doudna, and D. R. Liu (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31: 839–843.

    CAS  Article  Google Scholar 

  27. 27.

    Sakuma, T., A. Nishikawa, S. Kume, K. Chayama, and T. Yamamoto (2014) Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sci. Rep. 4: 5400.

  28. 28.

    Hsu, P. D., D. A. Scott, J. A. Weinstein, F. A. Ran, S. Konermann, V. Agarwala, Y. Li, E. J. Fine, X. Wu, O. Shalem, T. J. Cradick, L. A. Marraffini, G. Bao, and F. Zhang (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31: 827–832.

    CAS  Article  Google Scholar 

  29. 29.

    Kim, S., D. Kim, S. W. Cho, J. Kim, and J. S. Kim (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24: 1012–1019.

    CAS  Article  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Byung-Kwan Cho.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Shin, J., Lee, N., Song, Y. et al. Efficient CRISPR/Cas9-mediated multiplex genome editing in CHO cells via high-level sgRNA-Cas9 complex. Biotechnol Bioproc E 20, 825–833 (2015). https://doi.org/10.1007/s12257-015-0233-7

Download citation

Keywords

  • genome editing
  • CRISPR/Cas9
  • Chinese hamster ovary (CHO) cells
  • iterative transfection