Erythropoiesis pp 245-257 | Cite as

Genome Editing of Erythroid Cell Culture Model Systems

  • Jinfen J. Yik
  • Merlin Crossley
  • Kate G. R. Quinlan
Part of the Methods in Molecular Biology book series (MIMB, volume 1698)


Genome editing to introduce specific mutations or to knock out genes in model cell systems has become an efficient platform for research in the fields of molecular biology, genetics, and cell biology. With recent rapid improvements in genome editing techniques, bench-top manipulation of the genome in cell culture has become progressively easier. The application of this knowledge to erythroid cell culture systems now allows the rapid analysis of the downstream effects of virtually any engineered gene disruption or modification in cell systems. Here, we describe a CRISPR/Cas9-based approach to making genomic modifications in erythroid lineage cells which we have successfully used in both murine (MEL) and human (K562) erythroleukaemia immortalized cell lines.

Key words

Genome editing Genome engineering CRISPR/Cas9 Erythrocyte MEL K562 



This work has been supported by funding from the Australian Research Council and the National Health and Medical Research Council to M.C.


  1. 1.
    WHO|Sickle-cell disease and other haemoglobin disorders. Accessed 12 Dec 2015
  2. 2.
    Thein SL, Menzel S, Lathrop M, Garner C (2009) Control of fetal hemoglobin: new insights emerging from genomics and clinical implications. Hum Mol Genet 18:R216–R223CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Hemminger J (2012) Identification and reporting of common hemoglobin disorders: a review. Ibnosina J Med Biomed Sci 4:8–10Google Scholar
  4. 4.
    Bauer DE, Orkin SH (2011) Update on fetal hemoglobin gene regulation in hemoglobinopathies. Curr Opin Pediatr 23:1–8CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Bauer D, Kamran S, Orkin S (2015) Reawakening fetal hemoglobin: prospects for new therapies for the B-globin disorders. Blood 125:1061–1072CrossRefGoogle Scholar
  6. 6.
    Bank A (2006) Regulation of human fetal hemoglobin: new players, new complexities. Blood 107:435–443CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Wilber A, Nienhuis AW, Persons DA, Dc W (2013) Transcriptional regulation of fetal to adult hemoglobin switching: new therapeutic opportunities. Blood 117:3945–3953. doi: 10.1182/blood-2010-11-316893 CrossRefGoogle Scholar
  8. 8.
    Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–822CrossRefPubMedGoogle Scholar
  9. 9.
    Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32:347–355CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Richardson CD, Ray GJ, DeWitt MA et al (2016) Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol 34:339–344CrossRefPubMedGoogle Scholar
  12. 12.
    Masuda T, Wang X, Maeda M et al (2016) Transcription factors LRF and BCL11A independently repress expression of fetal hemoglobin. Science 351:285–289CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Domcke S, Bardet AF, Adrian Ginno P et al (2015) Competition between DNA methylation and transcription factors determines binding of NRF1. Nature 528:575–579CrossRefPubMedGoogle Scholar
  14. 14.
    Paquet D, Kwart D, Chen A et al (2016) Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533:125–129CrossRefPubMedGoogle Scholar
  15. 15.
    Komor AC, Kim YB, Packer MS et al (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 61:5985–5991Google Scholar
  16. 16.
    Wienert B, Funnell APW, Norton LJ et al (2015) Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin. Nat Commun 6:7085CrossRefPubMedGoogle Scholar
  17. 17.
    Kleinstiver BP, Pattanayak V, Prew MS et al (2016) High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529:490–495CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Finotti A, Breda L, Lederer CW et al (2015) Recent trends in the gene therapy of β-thalassemia. J Blood Med 6:69–85PubMedPubMedCentralGoogle Scholar
  19. 19.
    Puthenveetil G, Scholes J, Carbonell D et al (2004) Successful correction of the human β-thalassemia major phenotype using a lentiviral vector. Blood 104:3445–3453CrossRefPubMedGoogle Scholar
  20. 20.
    DeWitt M, Magis W, Bray NL, et al (2016) Efficient correction of the sickle mutation in human hematopoietic stem cells using a Cas9 ribonucleoprotein complex. bioRxiv 036236Google Scholar
  21. 21.
    Chang JC, Ye L, Kan YW (2006) Correction of the sickle cell mutation in embryonic stem cells. Proc Natl Acad Sci U S A 103:1036–1040CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Ran F, Hsu P, Wright J, Agarwala V (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Ayyadevara S, Thaden JJ, Shmookler Reis RJ (2000) Discrimination of primer 3′-nucleotide mismatch by taq DNA polymerase during polymerase chain reaction. Anal Biochem 284:11–18CrossRefPubMedGoogle Scholar
  24. 24.
    Chu VT, Weber T, Wefers B et al (2015) Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol 33:543–548CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Jinfen J. Yik
    • 1
  • Merlin Crossley
    • 1
  • Kate G. R. Quinlan
    • 1
  1. 1.School of Biotechnology and Biomolecular SciencesUniversity of New South WalesSydneyAustralia

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