Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool
Open accessAbstract
Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site‐specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome‐editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user‐friendly bioinformatics tool, named “CRISPy” for rapid identification of sgRNA target sequences in the CHO‐K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off‐target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells. Biotechnol. Bioeng. 2014; 111: 1604–1616. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Disruption of the fucosyltransferase in these cell lines completely abolished the fucosylation on the Fc domain of IgG.
“Disruption of the fucosyltransferase in these cell lines completely abolished the fucosylation on the Fc domain of IgG.”
From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Ronda et al., 2014
The Results section (‘Homozygous Knockout of FUT8 in CHO Cells Generated by CRISPR’; page unavailable) identifies FUT8 as the enzyme that adds fucose to CHO-produced IgG1 and reports FUT8 disruption. But its measurements concern cell-surface fucose and FUT8 indels, not fucosylation of the IgG Fc domain; moreover, 98.6% of selected cells, not all cells, were lectin-negative. It does not establish complete abolition of IgG Fc fucosylation in these cells. (LLM, conf=0.86)
Experimental — accuracy not yet measuredBoth ZFN and CRISPR/Cas9 approaches were also used for FUT8 gene-specific knockout.
“Recently, both ZFN and a CRISPR/Cas9 approaches were also used for FUT8 gene-specific knockout (Malphettes et al., 2010;Ronda et al., 2014).”
From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Ronda et al., 2014
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
No citation lists for this paper
No citation lists for this paper yet — checked again daily.
References 47 · Cited by 195
Research works printed but not linked: 37
- Bassett AR Tibbit C Ponting CP Liu J-L Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system Cell Rep 2013 4 220 228 23827738 PMC3714591
- Carroll D A CRISPR approach to gene targeting Mol Ther 2012 20 1658 1660 22945229 PMC3437577
- Chang N Sun C Gao L Zhu D Xu X Zhu X Xiong J-W Xi JJ Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos Cell Res 2013 23 465 472 23528705 PMC3616424
- Cho SW Kim S Kim JM Kim J-S Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease Nat Biotechnol 2013 31 230 232 23360966
- Cock PJA Antao T Chang JT Chapman BA Cox CJ Dalke A Friedberg I Hamelryck T Kauff F Wilczynski B de Hoon MJL Biopython: Freely available Python tools for computational molecular biology and bioinformatics Bioinformatics 2009 25 1422 1423 19304878 PMC2682512
- Cong L Ran FA Cox D Lin S Barretto R Habib N Hsu PD Wu X Jiang W Marraffini LA Zhang F Multiplex genome engineering using CRISPR/Cas systems Science 2013 339 819 823 23287718 PMC3795411
- Cost GJ Freyvert Y Vafiadis A Santiago Y Miller JC Rebar E Collingwood TN Snowden A Gregory PD BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cells Biotechnol Bioeng 2010 105 330 340 19777580
- DiCarlo JE Norville JE Mali P Rios X Aach J Church GM Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems Nucleic Acids Res 2013 41 4336 4343 23460208 PMC3627607
- Galetto R Duchateau P Pâques F Targeted approaches for gene therapy and the emergence of engineered meganucleases Expert Opin Biol Ther 2009 9 1289 1303 19689185
- Gratz SJ Cummings AM Nguyen JN Hamm DC Donohue LK Harrison JW O'Connor-Giles KM Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease Genetics 2013 194 1029 1035 23709638 PMC3730909
- Hammond S Kaplarevic M Borth N Betenbaugh MJ Lee KH Chinese hamster genome database Biotechnol Bioeng 2012 109 1353 1356 An online resource for the CHO community at http://www.chogenome.org 22105744
- Hansen BG Sun XE Genee HJ Kaas CS Nielsen JB Mortensen UH Frisvad JC Hedstrom L Adaptive evolution of drug targets in producer and non-producer organisms Biochem J 2012 441 219 226 21916847 PMC4636733
- Jiang W Zhou H Bi H Fromm M Yang B Weeks DP Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice Nucleic Acids Res 2013 41 1 12 23999092 PMC3814374
- Jinek M Chylinski K Fonfara I Hauer M Doudna JA Charpentier E A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity Science 2012 337 816 821 22745249 PMC6286148
- Jinek M East A Cheng A Lin S Ma E Doudna J RNA-programmed genome editing in human cells eLife 2013 2 1 9 PMC3557905 23386978
- Kildegaard HF Baycin-Hizal D Lewis NE Betenbaugh MJ The emerging CHO systems biology era: Harnessing the 'omics revolution for biotechnology Curr Opin Biotechnol 2013 24 1 6 23523260
- Lewis NE Liu X Li Y Nagarajan H Yerganian G O'Brien E Bordbar A Roth AM Rosenbloom J Bian C Xie M Chen W Li N Baycin-Hizal D Latif H Forster J Betenbaugh MJ Famili I Xu X Wang J Palsson BO Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome Nat Biotechnol 2013 31 759 765 23873082
- Ma M Ye AY Zheng W Kong L A guide RNA sequence design platform for the CRISPR/Cas9 system for model organism genomes BioMed Res Int 2013 2013 270805 24199189 PMC3809372
- Mali P Yang L Esvelt KM Aach J Guell M DiCarlo JE Norville JE Church GM RNA-guided human genome engineering via Cas9 Science 2013 339 823 826 23287722 PMC3712628
- Malphettes L Freyvert Y Chang J Liu P-Q Chan E Miller JC Zhou Z Nguyen T Tsai C Snowden AW Collingwood TN Gregory PD Cost GJ Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies Biotechnol Bioeng 2010 106 774 783 20564614
- Miller JC Tan S Qiao G Barlow K Wang J Xia DF Meng X Paschon DE Leung E Hinkley SJ Dulay GP Hua KL Ankoudinova I Cost GJ Urnov FD Zhang HS Holmes MC Zhang L Gregory PD Rebar EJ A TALE nuclease architecture for efficient genome editing Nat Biotechnol 2011 29 143 148 21179091
- Miyoshi E Noda K Yamaguchi Y Inoue S Ikeda Y Wang W Ko JH Uozumi N Li W Taniguchi N The alpha1-6-fucosyltransferase gene and its biological significance Biochem Biophys Acta 1999 1473 1 9 20 10580126
- Mori K Kuni-Kamochi R Yamane-Ohnuki N Wakitani M Yamano K Imai H Kanda Y Niwa R Iida S Uchida K Shitara K Satoh M Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA Biotechnol Bioeng 2004 88 901 908 15515168
- Niwa R Shoji-hosaka E Sakurada M Defucosylated chimeric anti-CC chemokine receptor 4 IgG1 with enhanced antibody-dependent cellular cytotoxicity shows potent therapeutic activity to T-cell leukemia and lymphoma Cancer Res 2004 64 2127 2133 15026353
- Nour-Eldin HH Hansen BG Nørholm MHH Jensen JK Halkier BA Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments Nucleic Acids Res 2006 34 e122 17000637 PMC1635280
- Nørholm M A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering BMC Biotechnol 2010 10 21 20233396 PMC2847956
- Pennisi E The CRISPR craze Science 2013 341 833 836 23970676
- Santiago Y Chan E Liu P-Q Orlando S Zhang L Urnov FD Holmes MC Guschin D Waite A Miller JC Rebar EJ Gregory PD Klug A Collingwood TN Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases Proc Natl Acad Sci USA 2008 105 5809 5814 18359850 PMC2299223
- Sedivy JM Sharp PA Positive genetic selection for gene disruption in mammalian cells by homologous recombination Proc Natl Acad Sci USA 1989 86 227 231 2536156 PMC286437
- Shields RL Lai J Keck R O'Connell LY Hong K Meng YG Weikert SH Presta LG Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity J Biol Chem 2002 277 26733 26740 11986321
- Steentoft C Vakhrushev SY Vester-Christensen MB Schjoldager KT Kong Y Bennett EP Mandel U Wandall H Levery SB Clausen H Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines Nat Methods 2011 8 977 982 21983924
- Waaijers S Portegijs V Kerver J Lemmens BB Tijsterman G van den Heuvel S Boxem M CRISPR/Cas9-targeted mutagenesis in C. elegans Genetics 2013 195 1187 1191 23979586 PMC3813849
- Wang T Wei JJ Sabatini DM Lander ES Genetic screens in human cells using the CRISPR-Cas9 system Science 2014 343 80 84 24336569 PMC3972032
- Wilson JR Williams D Schachter H The control of glycoprotein synthesis: N-acetylglucosamine linkage to a mannose residue as a signal for the attachment of l-fucose to the asparagine-linked N-acetylglucosamine residue of glycopeptide from alpha1-acid glycoprotein Biochem Biophys Res Commun 1976 72 909 916 985526
- Yamane-Ohnuki N Kinoshita S Inoue-Urakubo M Kusunoki M Iida S Nakano R Wakitani M Niwa R Sakurada M Uchida K Shitara K Satoh M 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 2004 87 614 622 15352059
- Yang H Wang H Shivalila CS Cheng AW Shi L Jaenisch R One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering Cell 2013 154 1370 1379 23992847 PMC3961003
- Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 2012 13 134 22708584 PMC3412702
- Authors
- Ronda, C., Pedersen, L. E., Hansen, H. G., Kallehauge, T. B., Betenbaugh, M. J., Nielsen, A. T., & Kildegaard, H. F.
- Affiliations
- Novo Nordisk Foundation (DK)
- Johns Hopkins University (US)
- Corresponding author
- Kildegaard, Helene Faustrup
- ORCID
- Venue
- Biotechnology and Bioengineering
- Publisher
- Wiley
- Published
- 2014-05-22
- Volume
- 111
- Issue
- 8
- Pages
- 1604-1616
- ISSN
- 0006-3592 (ISSN-L)
- 1097-0290
- PMC
- PMC4312910
- OpenAlex
- W2130005269
- Cited by
- 195
- References
- 47
- Citations by year
- 2026: 11 · 2025: 11 · 2024: 9 · 2023: 14 · 2022: 15 · 2021: 12 · 2020: 14 · 2019: 24 · 2018: 16 · 2017: 22 · 2016: 14 · 2015: 26 · 2014: 7
- FWCI
- 9.43
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Biotechnology and Bioengineering (publishedVersion, cc-by-nc)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- Technical University of Denmark, DTU Orbit (Technical University of Denmark, DTU) (submittedVersion)
- Funders
- The Novo Nordisk Foundation
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
- Keywords
- CRISPR/Cas9, CHO cells, genome editing, Cosmc, FUT8, glycosylation, indel frequency, frameshift mutation, off-target sites, biopharmaceutical manufacturing
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Animals, Base Sequence, Cricetulus, Endonucleases, Fucosyltransferases, Cricetinae, Molecular Sequence Data, CHO Cells, Genome, RNA Editing, Molecular Chaperones, Internet, Genomics, INDEL Mutation, Gene Knockout Techniques, High-Throughput Nucleotide Sequencing, Clustered Regularly Interspaced Short Palindromic Repeats
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
