Subtle Gene Modification in Mouse ES Cells: Evidence for Incorporation of Unmodified Oligonucleotides Without Induction of DNA Damage

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2010 Jul 6

PMID 20601408

Citations 21

Authors

Marieke Aarts

Hein Te Riele

Affiliations

Soon will be listed here.

Abstract

Gene targeting by single-stranded oligodeoxyribonucleotides (ssODNs) is a promising tool for site-specific gene modification in mouse embryonic stem cells (ESCs). We have developed an ESC line carrying a mutant EGFP reporter gene to monitor gene correction events shortly after exposure to ssODNs. We used this system to compare the appearance and fate of cells corrected by sense or anti-sense ssODNs. The slower appearance of green fluorescent cells with sense ssODNs as compared to anti-sense ssODNs is consistent with physical incorporation of the ssODN into the genome. Thus, the supremacy of anti-sense ssODNs, previously reported by others, may be an artefact of early readout of the EGFP reporter. Importantly, gene correction by unmodified ssODNs only mildly affected the viability of targeted cells and did not induce genomic DNA double-stranded breaks (DSBs). In contrast, ssODNs that were end-protected by phosphorothioate (PTO) linkages caused increased H2AX phosphorylation and impaired cell cycle progression in both corrected and non-corrected cells due to induction of genomic DSBs. Our results demonstrate that the use of unmodified rather than PTO end-protected ssODNs allows stable gene modification without compromising the genomic integrity of the cell, which is crucial for application of ssODN-mediated gene targeting in (embryonic) stem cells.

Citing Articles

CRISPR/Cas9-Mediated Introduction of Specific Heterozygous Mutations in Human Induced Pluripotent Stem Cells.

Brandao K, Grandela C, Yiangou L, Mummery C, Davis R Methods Mol Biol. 2021; 2454:531-557.

PMID: 33755904 PMC: 7612905. DOI: 10.1007/7651_2021_368.

Extensive trimming of short single-stranded DNA oligonucleotides during replication-coupled gene editing in mammalian cells.

van Ravesteyn T, Arranz Dols M, Pieters W, Dekker M, Te Riele H PLoS Genet. 2020; 16(10):e1009041.

PMID: 33119594 PMC: 7595315. DOI: 10.1371/journal.pgen.1009041.

Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates.

Romero Z, Lomova A, Said S, Miggelbrink A, Kuo C, Campo-Fernandez B Mol Ther. 2019; 27(8):1389-1406.

PMID: 31178391 PMC: 6697408. DOI: 10.1016/j.ymthe.2019.05.014.

Herpes ICP8 protein stimulates homologous recombination in human cells.

Valledor M, Myers R, Schiller P PLoS One. 2018; 13(8):e0200955.

PMID: 30110337 PMC: 6093641. DOI: 10.1371/journal.pone.0200955.

Easy quantification of template-directed CRISPR/Cas9 editing.

Brinkman E, Kousholt A, Harmsen T, Leemans C, Chen T, Jonkers J Nucleic Acids Res. 2018; 46(10):e58.

PMID: 29538768 PMC: 6007333. DOI: 10.1093/nar/gky164.

References

Engstrom J, Kmiec E . DNA replication, cell cycle progression and the targeted gene repair reaction. Cell Cycle. 2008; 7(10):1402-14. DOI: 10.4161/cc.7.10.5826. View

Dekker M, Brouwers C, Aarts M, van der Torre J, de Vries S, van de Vrugt H . Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3. Gene Ther. 2006; 13(8):686-94. DOI: 10.1038/sj.gt.3302689. View

Murphy B, Moayedpardazi H, Gewirtz A, Diamond S, Pierce E . Delivery and mechanistic considerations for the production of knock-in mice by single-stranded oligonucleotide gene targeting. Gene Ther. 2006; 14(4):304-15. DOI: 10.1038/sj.gt.3302866. View

Brachman E, Kmiec E . Gene repair in mammalian cells is stimulated by the elongation of S phase and transient stalling of replication forks. DNA Repair (Amst). 2005; 4(4):445-57. DOI: 10.1016/j.dnarep.2004.11.007. View

Bertoni C, Rustagi A, Rando T . Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides. Nucleic Acids Res. 2009; 37(22):7468-82. PMC: 2794159. DOI: 10.1093/nar/gkp757. View

Olsen P, Solhaug A, Booth J, Gelazauskaite M, Krauss S . Cellular responses to targeted genomic sequence modification using single-stranded oligonucleotides and zinc-finger nucleases. DNA Repair (Amst). 2008; 8(3):298-308. DOI: 10.1016/j.dnarep.2008.11.011. View

Bonner M, Kmiec E . DNA breakage associated with targeted gene alteration directed by DNA oligonucleotides. Mutat Res. 2009; 669(1-2):85-94. PMC: 2749079. DOI: 10.1016/j.mrfmmm.2009.05.004. View

Rosenkranz H, Levy J . HYDROXYUREA: A SPECIFIC INHIBITOR OF DEOXYRIBONUCLEIC ACID SYNTHESIS. Biochim Biophys Acta. 1965; 95:181-3. DOI: 10.1016/0005-2787(65)90225-x. View

Wu X, Xin L, Yin W, Shang X, Lu L, Watt R . Increased efficiency of oligonucleotide-mediated gene repair through slowing replication fork progression. Proc Natl Acad Sci U S A. 2005; 102(7):2508-13. PMC: 548982. DOI: 10.1073/pnas.0406991102. View

10.

Li X, Zhao X, Fang Y, Jiang X, Duong T, Fan C . Generation of destabilized green fluorescent protein as a transcription reporter. J Biol Chem. 1998; 273(52):34970-5. DOI: 10.1074/jbc.273.52.34970. View

11.

Paull T, Rogakou E, Yamazaki V, Kirchgessner C, Gellert M, Bonner W . A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol. 2000; 10(15):886-95. DOI: 10.1016/s0960-9822(00)00610-2. View

12.

Igoucheva O, Alexeev V, Anni H, Rubin E . Oligonucleotide-mediated gene targeting in human hepatocytes: implications of mismatch repair. Oligonucleotides. 2008; 18(2):111-22. PMC: 2966837. DOI: 10.1089/oli.2008.0120. View

13.

Olsen P, Randol M, Krauss S . Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides. Gene Ther. 2005; 12(6):546-51. DOI: 10.1038/sj.gt.3302454. View

14.

Olsen P, Randol M, Luna L, Brown T, Krauss S . Genomic sequence correction by single-stranded DNA oligonucleotides: role of DNA synthesis and chemical modifications of the oligonucleotide ends. J Gene Med. 2005; 7(12):1534-44. DOI: 10.1002/jgm.804. View

15.

Aarts M, Te Riele H . Parameters of oligonucleotide-mediated gene modification in mouse ES cells. J Cell Mol Med. 2009; 14(6B):1657-67. PMC: 3829028. DOI: 10.1111/j.1582-4934.2009.00847.x. View

16.

Ferrara L, Kmiec E . Targeted gene repair activates Chk1 and Chk2 and stalls replication in corrected cells. DNA Repair (Amst). 2006; 5(4):422-31. DOI: 10.1016/j.dnarep.2005.11.009. View

17.

Papaioannou I, Disterer P, Owen J . Use of internally nuclease-protected single-strand DNA oligonucleotides and silencing of the mismatch repair protein, MSH2, enhances the replication of corrected cells following gene editing. J Gene Med. 2009; 11(3):267-74. DOI: 10.1002/jgm.1296. View

18.

Aarts M, Dekker M, De Vries S, van der Wal A, Te Riele H . Generation of a mouse mutant by oligonucleotide-mediated gene modification in ES cells. Nucleic Acids Res. 2006; 34(21):e147. PMC: 1669774. DOI: 10.1093/nar/gkl896. View

19.

Radecke S, Radecke F, Peter I, Schwarz K . Physical incorporation of a single-stranded oligodeoxynucleotide during targeted repair of a human chromosomal locus. J Gene Med. 2005; 8(2):217-28. DOI: 10.1002/jgm.828. View

20.

Andrieu-Soler C, Casas M, Faussat A, Gandolphe C, Doat M, Tempe D . Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs. Nucleic Acids Res. 2005; 33(12):3733-42. PMC: 1174897. DOI: 10.1093/nar/gki686. View