High throughput, efficacious gene editing & genome surveillance in Chinese hamster ovary cells
Autoři:
S. C. Huhn aff001; Y. Ou aff001; A. Kumar aff001; R. Liu aff001; Z. Du aff001
Působiště autorů:
Cell Line Development, Merck & Co., Inc., Kenilworth, NJ, United States of America
aff001
Vyšlo v časopise:
PLoS ONE 14(12)
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pone.0218653
Souhrn
Chinese hamster ovary (CHO) cells are a common tool utilized in bioproduction and directed genome engineering of CHO cells is of great interest to enhance recombinant cell lines. Until recently, this focus has been challenged by a lack of efficacious, high throughput, and low-cost gene editing modalities and screening methods. In this work, we demonstrate an improved method for gene editing in CHO cells using CRISPR RNPs and characterize the endpoints of Cas9 and ZFN mediated genetic engineering. Furthermore, we validate sequence decomposition as a cost effective, rapid, and accurate method for assessing mutants and eliminating non-clonal CHO populations using only capillary sequencing.
Klíčová slova:
Polymerase chain reaction – CRISPR – CHO cells – Guide RNA – Gene pool – Tides – Zinc finger nucleases
Zdroje
1. Stolfa G, Smonskey MT, Boniface R, Hachmann AB, Gulde P, Joshi AD, et al. CHO-Omics Review: The Impact of Current and Emerging Technologies on Chinese Hamster Ovary Based Bioproduction. Biotechnol J. 2018;13(3):e1700227. Epub 2017/10/27. doi: 10.1002/biot.201700227 29072373.
2. Wang W, Zheng W, Hu F, He X, Wu D, Zhang W, et al. Enhanced Biosynthesis Performance of Heterologous Proteins in CHO-K1 Cells Using CRISPR-Cas9. ACS Synth Biol. 2018;7(5):1259–68. Epub 2018/04/24. doi: 10.1021/acssynbio.7b00375 29683658.
3. Lewis NE, Liu X, Li Y, Nagarajan H, Yerganian G, O'Brien E, et al. Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome. Nat Biotechnol. 2013;31(8):759–65. Epub 2013/07/23. doi: 10.1038/nbt.2624 23873082.
4. Tang D, Subramanian J, Haley B, Baker J, Luo L, Hsu W, et al. Pyruvate Kinase Muscle-1 Expression Appears to Drive Lactogenic Behavior in CHO Cell Lines, Triggering Lower Viability and Productivity: A Case Study. Biotechnol J. 2019;14(4):e1800332. Epub 2018/09/05. doi: 10.1002/biot.201800332 30179303.
5. Raab N, Mathias S, Alt K, Handrick R, Fischer S, Schmieder V, et al. CRISPR/Cas9-mediated knockout of microRNA-744 improves antibody titer of CHO production cell lines. Biotechnol J. 2019:e1800477. Epub 2019/02/26. doi: 10.1002/biot.201800477 30802343.
6. McVey D, Aronov M, Rizzi G, Cowan A, Scott C, Megill J, et al. CHO cells knocked out for TSC2 display an improved productivity of antibodies under fed batch conditions. Biotechnol Bioeng. 2016;113(9):1942–52. Epub 2016/02/19. doi: 10.1002/bit.25951 26888596.
7. Bauer DE, Canver MC, Orkin SH. Generation of genomic deletions in mammalian cell lines via CRISPR/Cas9. J Vis Exp. 2015;(95):e52118. Epub 2014/12/31. doi: 10.3791/52118 25549070; PubMed Central PMCID: PMC4279820.
8. Amann T, Hansen AH, Kol S, Lee GM, Andersen MR, Kildegaard HF. CRISPR/Cas9-Multiplexed Editing of Chinese Hamster Ovary B4Gal-T1, 2, 3, and 4 Tailors N-Glycan Profiles of Therapeutics and Secreted Host Cell Proteins. Biotechnol J. 2018;13(10):e1800111. Epub 2018/06/05. doi: 10.1002/biot.201800111 29862652.
9. Donohoue PD, Barrangou R, May AP. Advances in Industrial Biotechnology Using CRISPR-Cas Systems. Trends Biotechnol. 2018;36(2):134–46. Epub 2017/08/06. doi: 10.1016/j.tibtech.2017.07.007 28778606.
10. Gaj T, Guo J, Kato Y, Sirk SJ, Barbas CF 3rd. Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat Methods. 2012;9(8):805–7. Epub 2012/07/04. doi: 10.1038/nmeth.2030 22751204; PubMed Central PMCID: PMC3424280.
11. Isalan M. Zinc-finger nucleases: how to play two good hands. Nat Methods. 2011;9(1):32–4. Epub 2011/12/30. doi: 10.1038/nmeth.1805 22205514.
12. Sentmanat MF, Peters ST, Florian CP, Connelly JP, Pruett-Miller SM. A Survey of Validation Strategies for CRISPR-Cas9 Editing. Sci Rep. 2018;8(1):888. Epub 2018/01/19. doi: 10.1038/s41598-018-19441-8 29343825; PubMed Central PMCID: PMC5772360.
13. Brinkman EK, Chen T, Amendola M, van Steensel B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic acids research. 2014;42(22):e168. Epub 2014/10/11. doi: 10.1093/nar/gku936 25300484; PubMed Central PMCID: PMC4267669.
14. TIDE: Easy quantitative assessment of genome editing experiments by sequence trace decomposition: Desktop Genetics; 2017. Available from: https://www.tide.deskgen.com.
15. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34(2):184–91. Epub 2016/01/19. doi: 10.1038/nbt.3437 26780180; PubMed Central PMCID: PMC4744125.
16. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154(6):1380–9. Epub 2013/09/03. doi: 10.1016/j.cell.2013.08.021 23992846; PubMed Central PMCID: PMC3856256.
17. Pinello L, Canver MC, Hoban MD, Orkin SH, Kohn DB, Bauer DE, et al. Analyzing CRISPR genome editing experiments with CRISPResso. Nature biotechnology. 2016;34(7):695–7. doi: 10.1038/nbt.3583 PMC5242601. 27404874
18. Fan L, Kadura I, Krebs LE, Hatfield CC, Shaw MM, Frye CC. Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells. Biotechnol Bioeng. 2012;109(4):1007–15. Epub 2011/11/10. doi: 10.1002/bit.24365 22068567.
19. Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol. 2015;208:44–53. Epub 2015/05/25. S0168-1656(15)00200-X [pii] doi: 10.1016/j.jbiotec.2015.04.024 26003884.
20. Kim S, Kim D, Cho SW, Kim J, Kim JS. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 2014;24(6):1012–9. Epub 2014/04/04. gr.171322.113 [pii] doi: 10.1101/gr.171322.113 24696461; PubMed Central PMCID: PMC4032847.
21. Frye C, Deshpande R, Estes S, Francissen K, Joly J, Lubiniecki A, et al. Industry view on the relative importance of "clonality" of biopharmaceutical-producing cell lines. Biologicals. 2016;44(2):117–22. Epub 2016/02/08. S1045-1056(16)00002-6 [pii] doi: 10.1016/j.biologicals.2016.01.001 26852257.
22. Yang Z, Steentoft C, Hauge C, Hansen L, Thomsen AL, Niola F, et al. Fast and sensitive detection of indels induced by precise gene targeting. Nucleic acids research. 2015;43(9):e59. Epub 2015/03/11. doi: 10.1093/nar/gkv126 25753669; PubMed Central PMCID: PMC4482057.
23. Carroll D. Genome Engineering With Zinc-Finger Nucleases. Genetics. 2011;188(4):773–82. doi: 10.1534/genetics.111.131433 21828278; PubMed Central PMCID: PMC3176093.
24. Wah DA, Bitinaite J, Schildkraut I, Aggarwal AK. Structure of FokI has implications for DNA cleavage. Proc Natl Acad Sci U S A. 1998;95(18):10564–9. Epub 1998/09/02. doi: 10.1073/pnas.95.18.10564 9724743; PubMed Central PMCID: PMC27934.
Článok vyšiel v časopise
PLOS One
2019 Číslo 12
- Metamizol jako analgetikum první volby: kdy, pro koho, jak a proč?
- Nejasný stín na plicích – kazuistika
- Masturbační chování žen v ČR − dotazníková studie
- Těžké menstruační krvácení může značit poruchu krevní srážlivosti. Jaký management vyšetření a léčby je v takovém případě vhodný?
- Fixní kombinace paracetamol/kodein nabízí synergické analgetické účinky
Najčítanejšie v tomto čísle
- Methylsulfonylmethane increases osteogenesis and regulates the mineralization of the matrix by transglutaminase 2 in SHED cells
- Oregano powder reduces Streptococcus and increases SCFA concentration in a mixed bacterial culture assay
- The characteristic of patulous eustachian tube patients diagnosed by the JOS diagnostic criteria
- Parametric CAD modeling for open source scientific hardware: Comparing OpenSCAD and FreeCAD Python scripts