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Basiri M, Behmanesh M, Tahamtani Y, et al. The Convenience of Single Homology Arm Donor DNA and CRISPR/Cas9-Nickase for Targeted Insertion of Long DNA Fragment. Cell J. 2016;18(4):532-539doi: 10.22074/cellj.2016.4719.
Basiri, M., Behmanesh, M., Tahamtani, Y., Khalooghi, K., Moradmand, A., & Baharvand, H. (2017). The Convenience of Single Homology Arm Donor DNA and CRISPR/Cas9-Nickase for Targeted Insertion of Long DNA Fragment. Cell journal, 18(4), 532-539. https://doi.org/10.22074/cellj.2016.4719
Basiri, Mohsen, et al. "The Convenience of Single Homology Arm Donor DNA and CRISPR/Cas9-Nickase for Targeted Insertion of Long DNA Fragment." Cell journal vol. 18,4 (2017): 532-539. doi: https://doi.org/10.22074/cellj.2016.4719
Basiri M, Behmanesh M, Tahamtani Y, Khalooghi K, Moradmand A, Baharvand H. The Convenience of Single Homology Arm Donor DNA and CRISPR/Cas9-Nickase for Targeted Insertion of Long DNA Fragment. Cell J. 2017;18(4):532-539. doi: 10.22074/cellj.2016.4719. Epub 2016 Sep 26. PMID: 28042537; PMCID: PMC5086331.
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Cell J. 2017;18(4):532-539. doi: 10.22074/cellj.2016.4719. Epub 2016 Sep 26.

The Convenience of Single Homology Arm Donor DNA and CRISPR/Cas9-Nickase for Targeted Insertion of Long DNA Fragment.

Cell journal

Mohsen Basiri, Mehrdad Behmanesh, Yaser Tahamtani, Keynoosh Khalooghi, Azadeh Moradmand, Hossein Baharvand

Affiliations

  1. Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran; Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
  2. Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
  3. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
  4. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Developmental Biology, University of Science and Culture, Tehran, Iran.

PMID: 28042537 PMCID: PMC5086331 DOI: 10.22074/cellj.2016.4719

Abstract

OBJECTIVE: CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid.

MATERIALS AND METHODS: In this experimental study, single homology arm donor was applied along with a single guide RNA (sgRNA) specific to the homology region, and either Cas9 or its mutant nickase variant (Cas9n). Using

RESULTS: Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of

CONCLUSION: While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies.

Keywords: CRISPR-Cas Systems; Embryonic Stem Cells; Gene Targeting; Pdx1

References

  1. Stem Cell Rev. 2012 Jun;8(2):472-81 - PubMed
  2. Stem Cell Rev. 2014 Feb;10(1):16-30 - PubMed
  3. Cell. 2013 Sep 12;154(6):1380-9 - PubMed
  4. Nat Biotechnol. 2013 Sep;31(9):833-8 - PubMed
  5. Biotechniques. 2004 Oct;37(4):610-3 - PubMed
  6. PLoS One. 2014 Aug 28;9(8):e105779 - PubMed
  7. Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):5172-7 - PubMed
  8. Annu Rev Genet. 2006;40:363-83 - PubMed
  9. Transgenic Res. 2002 Feb;11(1):43-8 - PubMed
  10. Cell J. 2013 Summer;15(2):190-7 - PubMed
  11. Oncogene. 2007 Dec 10;26(56):7731-40 - PubMed
  12. Protein Cell. 2014 Apr;5(4):258-60 - PubMed
  13. Exp Hematol. 2002 Jun;30(6):503-12 - PubMed
  14. Physiol Genomics. 2008 Aug 15;34(3):225-38 - PubMed
  15. Nat Biotechnol. 2013 Sep;31(9):827-32 - PubMed
  16. Science. 2013 Feb 15;339(6121):819-23 - PubMed
  17. Science. 2013 Feb 15;339(6121):823-6 - PubMed
  18. Cell. 2013 Sep 12;154(6):1370-9 - PubMed
  19. Biochem Biophys Res Commun. 2014 Mar 21;445(4):791-4 - PubMed
  20. Nat Protoc. 2013 Nov;8(11):2281-308 - PubMed
  21. Cell Rep. 2014 Apr 10;7(1):293-305 - PubMed
  22. Mol Cell Biol. 1992 Jun;12(6):2464-74 - PubMed

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