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Leishmania pp 169–188Cite as

Gene Replacement by Homologous Recombination

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1971))

Abstract

While homologous recombination-based gene replacement is about to be supplanted by more modern approaches, it is still retaining usefulness for genes that prove to be poor targets for CRISPR/cas-based approaches. Homologous recombination has proven to be relatively robust to minor sequence mismatches between GOI-flanking sequences and the gene replacement constructs, and the faithfulness of recombination events is easily verified by whole-genome sequencing. Moreover, the availability of custom synthetic gene production by numerous service providers should allow for a relatively quick generation of null mutants without the need to introduce additional protein-coding genes beyond the selection markers.

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References

  1. Cruz A, Beverley SM (1990) Gene replacement in parasitic protozoa. Nature 348:171–173

    Article  CAS  Google Scholar 

  2. Cruz A, Coburn CM, Beverley SM (1991) Double targeted gene replacement for creating null mutants. Proc Natl Acad Sci U S A 88:7170–7174

    Article  CAS  Google Scholar 

  3. Sollelis L, Ghorbal M, MacPherson CR, Martins RM, Kuk N, Crobu L, Bastien P, Scherf A, Lopez-Rubio JJ, Sterkers Y (2015) First efficient CRISPR-Cas9-mediated genome editing in Leishmania parasites. Cell Microbiol 17(10):1405–1412. https://doi.org/10.1111/cmi.12456

    Article  CAS  PubMed  Google Scholar 

  4. Zhang WW, Matlashewski G (2015) CRISPR-Cas9-mediated genome editing in Leishmania donovani. MBio 6(4):e00861. https://doi.org/10.1128/mBio.00861-15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Duncan SM, Myburgh E, Philipon C, Brown E, Meissner M, Brewer J, Mottram JC (2016) Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation. Mol Microbiol 100(6):931–944. https://doi.org/10.1111/mmi.13375

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Beneke T, Madden R, Makin L, Valli J, Sunter J, Gluenz E (2017) A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids. R Soc Open Sci 4(5):170095. https://doi.org/10.1098/rsos.170095

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Martel D, Beneke T, Gluenz E, Spath GF, Rachidi N (2017) Characterisation of casein kinase 1.1 in Leishmania donovani using the CRISPR Cas9 toolkit. Biomed Res Int 2017:4635605. https://doi.org/10.1155/2017/4635605

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bifeld E, Chrobak M, Zander D, Schleicher U, Schonian G, Clos J (2015) Geographical sequence variation in the Leishmania major virulence factor P46. Infect Genet Evol 30:195–205. https://doi.org/10.1016/j.meegid.2014.12.029

    Article  PubMed  Google Scholar 

  9. Ubeda JM, Raymond F, Mukherjee A, Plourde M, Gingras H, Roy G, Lapointe A, Leprohon P, Papadopoulou B, Corbeil J, Ouellette M (2014) Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania. PLoS Biol 12(5):e1001868. https://doi.org/10.1371/journal.pbio.1001868

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Papadopoulou B, Dumas C (1997) Parameters controlling the rate of gene targeting frequency in the protozoan parasite Leishmania. Nucleic Acids Res 25(21):4278–4286

    Article  CAS  Google Scholar 

  11. Krobitsch S, Clos J (2000) Cross-species homologous recombination in Leishmania donovani reveals the sites of integration. Mol Biochem Parasitol 107:123–128

    Article  CAS  Google Scholar 

  12. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33(1):103–119

    Article  CAS  Google Scholar 

  13. Bartsch K, Hombach-Barrigah A, Clos J (2017) Hsp90 inhibitors radicicol and geldanamycin have opposing effects on Leishmania Aha1-dependent proliferation. Cell Stress Chaperones 22:729–742. https://doi.org/10.1007/s12192-017-0800-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ommen G, Lorenz S, Clos J (2009) One-step generation of double-allele gene replacement mutants in Leishmania donovani. Int J Parasitol 39(5):541–546

    Article  CAS  Google Scholar 

  15. Sambrook J, Russell DW (2001) Molecular Cloning, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  16. Schäfer C, Tejera Nevado P, Zander D, Clos J (2014) ARM58 overexpression reduces intracellular antimony concentration in Leishmania infantum. Antimicrob Agents Chemother 58:1565–1574. https://doi.org/10.1128/AAC.01881-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685

    Article  CAS  Google Scholar 

  18. Hombach A, Ommen G, MacDonald A, Clos J (2014) A small heat shock protein is essential for thermotolerance and intracellular survival of Leishmania donovani. J Cell Sci 127(Pt 21):4762–4773. https://doi.org/10.1242/jcs.157297

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tejera Nevado P, Bifeld E, Hohn K, Clos J (2016) A Telomeric cluster of antimony resistance genes on chromosome 34 of Leishmania infantum. Antimicrob Agents Chemother 60(9):5262–5275. https://doi.org/10.1128/AAC.00544-16

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We are grateful to laboratory alumni Andreas Hübel, Sylvia Krobitsch, Gabi Ommen, Katharina Bartsch, Eugenia Bifeld, and Antje Hombach for their contributions to the refinement of the homologous gene recombination strategy in the laboratory.

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Authors and Affiliations

  1. Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany

    Henner Zirpel & Joachim Clos

Authors
  1. Henner Zirpel
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  2. Joachim Clos
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Corresponding author

Correspondence to Joachim Clos .

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Editors and Affiliations

  1. Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany

    Joachim Clos

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Zirpel, H., Clos, J. (2019). Gene Replacement by Homologous Recombination. In: Clos, J. (eds) Leishmania. Methods in Molecular Biology, vol 1971. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9210-2_8

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  • DOI: https://doi.org/10.1007/978-1-4939-9210-2_8

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9209-6

  • Online ISBN: 978-1-4939-9210-2

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