Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Cellular and Molecular Biology Letters

Online
ISSN
1689-1392
See all formats and pricing
More options …
Volume 20, Issue 5

Issues

Homology arms of targeting vectors for gene insertions and CRISPR/Cas9 technology: size does not matter; quality control of targeted clones does

Silvia Petrezselyova
  • Institute of Molecular Genetics of the ASCR, v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Slavomir Kinsky
  • Institute of Molecular Genetics of the ASCR, v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Dominika Truban
  • Institute of Molecular Genetics of the ASCR, v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Radislav Sedlacek
  • Institute of Molecular Genetics of the ASCR, v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Ingo Burtscher
  • Institute of Stem Cell Research, Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), Ingolstädter Landstr. 1, 85764 Neuherberg, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Heiko Lickert
  • Corresponding author
  • Institute of Stem Cell Research, Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), Ingolstädter Landstr. 1, 85764 Neuherberg, Germany
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2016-03-05 | DOI: https://doi.org/10.1515/cmble-2015-0047

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology has brought rapid progress in mammalian genome editing (adding, disrupting or changing the sequence of specific sites) by increasing the frequency of targeted events. However, gene knock-in of DNA cassettes by homologous recombination still remains difficult due to the construction of targeting vectors possessing large homology arms (from 2 up to 5 kb). Here, we demonstrate that in mouse embryonic stem cells the combination of CRISPR/Cas9 technology and targeting vectors with short homology arms (~ 0.3 kb) provides sufficient specificity for insertion of fluorescent reporter cassettes into endogenous genes with similar efficiency as those with large conventional vectors. Importantly, we emphasize the necessity of thorough quality control of recombinant clones by combination of the PCR method, Southern hybridization assay and sequencing to exclude undesired mutations. In conclusion, our approach facilitates programmed integration of exogenous DNA sequences at a target locus and thus could serve as a basis for more sophisticated genome engineering approaches, such as generation of reporters and conditional knock-out alleles.

Keywords: CRISPR/Cas9; Genome editing; Reporter; Targeting vector; Homology arms; Embryonic stem cell

References

  • 1. Capecchi, M.R. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat. Rev. Genet. 6 (2005) 507-512.CrossrefGoogle Scholar

  • 2. Gaj, T., Gersbach, C.A. and Barbas C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31 (2013) 397-405.CrossrefGoogle Scholar

  • 3. Hasty, P., Rivera-Perez, J. and Bradley A. The length of homology required for gene targeting in embryonic stem cells. Mol. Cell Biol. 11 (1991) 5586-5591.CrossrefGoogle Scholar

  • 4. Deng, C. and Capecchi, M.R. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol. Cell Biol. 12 (1992) 3365-3371.CrossrefGoogle Scholar

  • 5. Shulman, M.J., Nissen, L. and Collins C. Homologous recombination in hybridoma cells: dependence on time and fragment length. Mol. Cell Biol. 10 (1990) 4466-4472.CrossrefGoogle Scholar

  • 6. Ran, F.A., Hsu, P.D., Lin, C.Y., Gootenberg, J.S., Konermann, S., Trevino, A.E., Scott, D.A., Inoue, A., Matoba, S., Zhang, Y. and Zhang, F. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154 (2013) 1380-1309.Web of ScienceGoogle Scholar

  • 7. Copeland, N.G., Jenkins, N.A. and Court, D.L. Recombineering: a powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2 (2001) 769-779.CrossrefGoogle Scholar

  • 8. Engert, S., Liao, W.P., Burtscher, I. and Lickert, H. Sox17-2A-iCre: a knock-in mouse line expressing Cre recombinase in endoderm and vascular endothelial cells. Genesis 47 (2009) 603-610.Web of ScienceGoogle Scholar

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

  • 10. Uetzmann, L., Burtscher, I. and Lickert, H. A mouse line expressing Foxa2- driven Cre recombinase in node, notochord, floorplate, and endoderm. Genesis 10 (2008) 515-522.CrossrefWeb of ScienceGoogle Scholar

  • 11. Hitz, C., Wurst, W. and Kuhn, R. Conditional brain-specific knockdown of MAPK using Cre/loxP regulated RNA interference. Nucleic Acids Res. 35 (2007) e90.CrossrefWeb of ScienceGoogle Scholar

  • 12. Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E. and Tsien, R.Y. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22 (2004) 1567-1572.CrossrefGoogle Scholar

  • 13. Li, K., Wang, G., Andersen, T., Zhou, P. and Pu, W.T. Optimization of genome engineering approaches with the CRISPR/Cas9 system. PLoS One 9 (2014) e105779.Google Scholar

  • 14. Hisano, Y., Sakuma, T., Nakade, S., Ohga, R., Ota, S., Okamoto, H., Yamamoto, T. and Kawahara, A. Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish. Sci. Rep. 5 (2015) 8841.CrossrefWeb of ScienceGoogle Scholar

  • 15. Paix, A., Wang, Y., Smith, H.E., Lee, C.Y., Calidas, D., Lu, T., Smith, J., Schmidt, H., Krause, M.W. and Seydoux, G. Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics 198 (2014) 1347-1356. Web of ScienceGoogle Scholar

About the article

Received: 2015-06-17

Accepted: 2015-10-15

Published Online: 2016-03-05

Published in Print: 2015-12-01


Citation Information: Cellular and Molecular Biology Letters, Volume 20, Issue 5, Pages 773–787, ISSN (Online) 1689-1392, DOI: https://doi.org/10.1515/cmble-2015-0047.

Export Citation

© University of Wroclaw, Poland. Copyright Clearance Center

Citing Articles

Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the “Citation Alert” on the top of this page.

[1]
Yibo Zhang, Zhiwei Zhang, and Wei Ge
Journal of Biological Chemistry, 2018, Volume 293, Number 17, Page 6611

Comments (0)

Please log in or register to comment.
Log in