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Precise and Scarless Insertion of Transposable Elements by Cas9-Mediated Genome Engineering

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Transposable Elements

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2607))

Abstract

Transposable element insertions can have broad effects on gene expression, ranging from new regulatory functions to pathogenic consequences by transplanting new cis-regulating elements or perturbing existing ones. Genetic manipulation of such DNA sequences can help decipher their mechanism of action. Here, we describe a CRISPR-Cas9-mediated two-step approach to precisely insert transposable elements into into the genome of cultured human cells, without scar or reporter gene. First, a double-selection cassette is inserted into the desired target locus. Once a clone containing a single copy of this cassette has been isolated, a second editing step is performed to exchange the double-selection cassette with a markerless transposable element sequence. More generally, this method can be used for knocking in any large insert without genetic markers.

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References

  1. Cordaux R, Batzer MA (2009) The impact of retrotransposons on human genome evolution. Nat Rev Genet 10:691–703. https://doi.org/10.1038/nrg2640

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kazazian HH, Wong C, Youssoufian H, Scott AF (1988) Haemophilia A resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man. Nature 332:164–166. https://doi.org/10.1038/332164a0

    Article  CAS  PubMed  Google Scholar 

  3. Hancks DC, Kazazian HH (2016) Roles for retrotransposon insertions in human disease. Mob DNA 7:9–28. https://doi.org/10.1186/s13100-016-0065-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Belancio VP, Hedges DJ, Deininger P (2006) LINE-1 RNA splicing and influences on mammalian gene expression. Nucleic Acids Res 34:1512–1521. https://doi.org/10.1093/nar/gkl027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Belancio VP, Roy-Engel AM, Deininger P (2008) The impact of multiple splice sites in human L1 elements. Gene 411:38–45. https://doi.org/10.1016/j.gene.2007.12.022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Rishishwar L, Wang L, Wang J et al (2018) Evidence for positive selection on recent human transposable element insertions. Gene 675:69–79. https://doi.org/10.1016/j.gene.2018.06.077

    Article  CAS  PubMed  Google Scholar 

  7. Pontis J, Planet E, Offner S et al (2019) Hominoid-specific transposable elements and KZFPs facilitate human embryonic genome activation and control transcription in naive human ESCs. Cell Stem Cell 24:724–735.e5. https://doi.org/10.1016/j.stem.2019.03.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Chuong EB, Elde NC, Feschotte C (2016) Regulatory activities of transposable elements: from conflicts to benefits. Nat Rev Genet 18:71. https://doi.org/10.1038/nrg.2016.139

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Fuentes DR, Swigut T, Wysocka J (2018) Systematic perturbation of retroviral LTRs reveals widespread long-range effects on human gene regulation. eLife 7:861. https://doi.org/10.7554/eLife.35989

    Article  Google Scholar 

  10. Todd CD, Deniz Ö, Taylor D, Branco MR (2019) Functional evaluation of transposable elements as enhancers in mouse embryonic and trophoblast stem cells. eLife 8:e44344. https://doi.org/10.7554/eLife.44344

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Chuong EB, Elde NC, Feschotte C (2016) Regulatory evolution of innate immunity through co-option of endogenous retroviruses. Science 351:1083–1087. https://doi.org/10.1126/science.aad5497

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Xiong F, Wang R, Lee J-H et al (2021) RNA m6A modification orchestrates a LINE-1-host interaction that facilitates retrotransposition and contributes to long gene vulnerability. Cell Res 31:861–885. https://doi.org/10.1038/s41422-021-00515-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Schwahn U, Lenzner S, Dong J et al (1998) Positional cloning of the gene for X-linked retinitis pigmentosa 2. Nat Genet 19:327–332. https://doi.org/10.1038/1214

    Article  CAS  PubMed  Google Scholar 

  14. Kimberland ML, Divoky V, Prchal J et al (1999) Full-length human L1 insertions retain the capacity for high frequency retrotransposition in cultured cells. Hum Mol Genet 8:1557–1560. https://doi.org/10.1093/hmg/8.8.1557

    Article  CAS  PubMed  Google Scholar 

  15. Badge RM, Alisch RS, Moran JV (2003) ATLAS: a system to selectively identify human-specific L1 insertions. Am J Hum Genet 72:823–838. https://doi.org/10.1086/373939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lackner DH, Carré A, Guzzardo PM et al (2015) A generic strategy for CRISPR-Cas9-mediated gene tagging. Nat Commun:1–7. https://doi.org/10.1038/ncomms10237

  17. Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826. https://doi.org/10.1126/science.1232033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Brinkman EK, Chen T, Amendola M, van Steensel B (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42:e168–e168. https://doi.org/10.1093/nar/gku936

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lin S, Staahl BT, Alla RK, Doudna JA (2014) Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3:1314. https://doi.org/10.7554/eLife.04766

    Article  Google Scholar 

  20. Liang X, Potter J, Kumar S et al (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol 208:44–53. https://doi.org/10.1016/j.jbiotec.2015.04.024

    Article  CAS  PubMed  Google Scholar 

  21. Zhang Y, Werling U, Edelmann W (2012) SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res 40:e55–e55. https://doi.org/10.1093/nar/gkr1288

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ryan JA (2008) Cell cloning by serial dilution in 96 well plates—protocol. In: Corning. https://www.corning.com/catalog/cls/documents/protocols/Single_cell_cloning_protocol.pdf. Accessed 1 Mar 2022

  23. Miyaoka Y, Chan AH, Judge LM et al (2014) Isolation of single-base genome-edited human iPS cells without antibiotic selection. Nat Methods 11:291–293. https://doi.org/10.1038/nmeth.2840

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Touraine RL, Ishii-Morita H, Ramsey WJ, Blaese RM (1998) The bystander effect in the HSVtk/ganciclovir system and its relationship to gap junctional communication. Gene Ther 5:1705–1711. https://doi.org/10.1038/sj.gt.3300784

    Article  CAS  PubMed  Google Scholar 

  25. Gentry BG, Im M, Boucher PD et al (2005) GCV phosphates are transferred between HeLa cells despite lack of bystander cytotoxicity. Gene Ther 12:1033–1041. https://doi.org/10.1038/sj.gt.3302487

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the French government, through the Agence Nationale de la Recherche (Idex UCAJEDI, ANR-15-IDEX-01; Labex SIGNALIFE, ANR-11-LABX-0028-01; ImpacTE, ANR-19-CE12-0032), the Fondation pour la Recherche Médicale (FRM, DEQ20180339170), Inserm (GOLD cross-cutting program on genomic variability), and CNRS (GDR 3546). We are grateful to IRCAN genomic and cytometry platforms, Genomed, and Cytomed. Equipment acquisition for IRCAN platforms was supported by FEDER, Région Provence Alpes Côte d’Azur, Canceropole PACA, Conseil Départemental 06, ITMO Cancer Aviesan (plan cancer) and Inserm.

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Correspondence to Aurélien J. Doucet or Gael Cristofari .

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Weber, V.M., Doucet, A.J., Cristofari, G. (2023). Precise and Scarless Insertion of Transposable Elements by Cas9-Mediated Genome Engineering. In: Branco, M.R., de Mendoza Soler, A. (eds) Transposable Elements. Methods in Molecular Biology, vol 2607. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2883-6_15

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  • DOI: https://doi.org/10.1007/978-1-0716-2883-6_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2882-9

  • Online ISBN: 978-1-0716-2883-6

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