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VCre/VloxP and SCre/SloxP as Reliable Site-Specific Recombination Systems for Genome Engineering

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Genome Editing in Animals

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2637))

Abstract

The Cre/loxP system is a versatile and powerful tool that has been used to develop many kinds of genetically modified mice, such as conditional knockout mice and mutant protein-expressing mice through the excision of a STOP cassette. However, while numerous in vivo and in vitro applications of the Cre/loxP system have been reported, it remains difficult to target at one time more than one set of recognition sites in an identical single cell in mice using the Cre/loxP system. To overcome this barrier, we developed two novel site-specific recombination systems called VCre/VloxP and SCre/SloxP. These systems allow multiple independent site-specific recombination, for example, multiple targeted deletions in the same cell at different times. In this chapter, I describe the features of VCre/VloxP and SCre/SloxP, practical protocols and tips on how to use them in genomic engineering applications, potential problems in their use, and how problems can be identified and solved.

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References

  1. Nagy A (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis (New York, NY: 2000) 26(2):99–109

    Article  CAS  Google Scholar 

  2. Kim H, Kim M, Im SK, Fang S (2018) Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes. Lab Anim Res 34(4):147–159. https://doi.org/10.5625/lar.2018.34.4.147

    Article  Google Scholar 

  3. Turan S, Zehe C, Kuehle J, Qiao J, Bode J (2013) Recombinase-mediated cassette exchange (RMCE) - a rapidly-expanding toolbox for targeted genomic modifications. Gene 515(1):1–27. https://doi.org/10.1016/j.gene.2012.11.016

    Article  CAS  Google Scholar 

  4. Cai D, Cohen KB, Luo T, Lichtman JW, Sanes JR (2013) Improved tools for the Brainbow toolbox. Nat Methods 10(6):540–547. https://doi.org/10.1038/nmeth.2450

    Article  CAS  Google Scholar 

  5. Schnütgen F, Doerflinger N, Calléja C, Wendling O, Chambon P, Ghyselinck NB (2003) A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat Biotechnol 21(5):562–565. https://doi.org/10.1038/nbt811

    Article  CAS  Google Scholar 

  6. Suzuki K, Mitsui K, Aizawa E, Hasegawa K, Kawase E, Yamagishi T, Shimizu Y, Suemori H, Nakatsuji N, Mitani K (2008) Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors. Proc Natl Acad Sci U S A 105(37):13781–13786. https://doi.org/10.1073/pnas.0806976105

    Article  Google Scholar 

  7. Suzuki E, Nakayama M (2011) VCre/VloxP and SCre/SloxP: new site-specific recombination systems for genome engineering. Nucleic Acids Res 39(8):e49. https://doi.org/10.1093/nar/gkq1280

    Article  CAS  Google Scholar 

  8. Yoshimura Y, Ida-Tanaka M, Hiramaki T, Goto M, Kamisako T, Eto T, Yagoto M, Kawai K, Takahashi T, Nakayama M, Ito M (2018) Novel reporter and deleter mouse strains generated using VCre/VloxP and SCre/SloxP systems, and their system specificity in mice. Transgenic Res 27(2):193–201. https://doi.org/10.1007/s11248-018-0067-0

    Article  CAS  Google Scholar 

  9. Kishimoto K, Nakayama M, Kinoshita M (2016) In vivo recombination efficiency of two site-specific recombination systems, VCre/VloxP and SCre/SloxP, in medaka (Oryzias latipes). Develop Growth Differ 58(6):516–521. https://doi.org/10.1111/dgd.12289

    Article  CAS  Google Scholar 

  10. Minorikawa S, Nakayama M (2011) Recombinase-mediated cassette exchange (RMCE) and BAC engineering via VCre/VloxP and SCre/SloxP systems. BioTechniques 50(4):235–246. https://doi.org/10.2144/000113649

    Article  CAS  Google Scholar 

  11. Khateb M, Fourier N, Barnea-Yizhar O, Ram S, Kovalev E, Azriel A, Rand U, Nakayama M, Hauser H, Gepstein L, Levi BZ (2016) The third intron of the interferon regulatory factor-8 is an initiator of repressed chromatin restricting its expression in non-immune cells. PLoS One 11(6):e0156812. https://doi.org/10.1371/journal.pone.0156812

    Article  CAS  Google Scholar 

  12. Lee G, Saito I (1998) Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216(1):55–65. https://doi.org/10.1016/s0378-1119(98)00325-4

    Article  CAS  Google Scholar 

  13. Guo F, Gopaul DN, van Duyne GD (1997) Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature 389(6646):40–46. https://doi.org/10.1038/37925

    Article  CAS  Google Scholar 

  14. Gibb B, Gupta K, Ghosh K, Sharp R, Chen J, Van Duyne GD (2010) Requirements for catalysis in the Cre recombinase active site. Nucleic Acids Res 38(17):5817–5832. https://doi.org/10.1093/nar/gkq384

    Article  CAS  Google Scholar 

  15. Kon A, Yamazaki S, Nannya Y, Kataoka K, Ota Y, Nakagawa MM, Yoshida K, Shiozawa Y, Morita M, Yoshizato T, Sanada M, Nakayama M, Koseki H, Nakauchi H, Ogawa S (2018) Physiological Srsf2 P95H expression causes impaired hematopoietic stem cell functions and aberrant RNA splicing in mice. Blood 131(6):621–635. https://doi.org/10.1182/blood-2017-01-762393

    Article  CAS  Google Scholar 

  16. Hasegawa Y, Ikeno M, Suzuki N, Nakayama M, Ohara O (2018) Improving the efficiency of gene insertion in a human artificial chromosome vector and its transfer in human-induced pluripotent stem cells. Biol Methods Protoc 3(1):bpy013. https://doi.org/10.1093/biomethods/bpy013

    Article  CAS  Google Scholar 

  17. Kim GB, Rincon Fernandez Pacheco D, Saxon D, Yang A, Sabet S, Dutra-Clarke M, Levy R, Watkins A, Park H, Abbasi Akhtar A, Linesch PW, Kobritz N, Chandra SS, Grausam K, Ayala-Sarmiento A, Molina J, Sedivakova K, Hoang K, Tsyporin J, Gareau DS, Filbin MG, Bannykh S, Santiskulvong C, Wang Y, Tang J, Suva ML, Chen B, Danielpour M, Breunig JJ (2019) Rapid generation of somatic mouse mosaics with locus-specific. Stably Integr Transgenic Elem Cell 179(1):251–267.e224. https://doi.org/10.1016/j.cell.2019.08.013

    Article  CAS  Google Scholar 

  18. Fenno LE, Mattis J, Ramakrishnan C, Hyun M, Lee SY, He M, Tucciarone J, Selimbeyoglu A, Berndt A, Grosenick L, Zalocusky KA, Bernstein H, Swanson H, Perry C, Diester I, Boyce FM, Bass CE, Neve R, Huang ZJ, Deisseroth K (2014) Targeting cells with single vectors using multiple-feature Boolean logic. Nat Methods 11(7):763–772. https://doi.org/10.1038/nmeth.2996

    Article  CAS  Google Scholar 

  19. Fenno LE, Ramakrishnan C, Kim YS, Evans KE, Lo M, Vesuna S, Inoue M, Cheung KYM, Yuen E, Pichamoorthy N, Hong ASO, Deisseroth K (2020) Comprehensive dual- and triple-feature intersectional single-vector delivery of diverse functional payloads to cells of behaving mammals. Neuron 107(5):836–853.e811. https://doi.org/10.1016/j.neuron.2020.06.003

    Article  CAS  Google Scholar 

  20. Weinberg BH, Pham NTH, Caraballo LD, Lozanoski T, Engel A, Bhatia S, Wong WW (2017) Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells. Nat Biotechnol 35(5):453–462. https://doi.org/10.1038/nbt.3805

    Article  CAS  Google Scholar 

  21. Weinberg BH, Cho JH, Agarwal Y, Pham NTH, Caraballo LD, Walkosz M, Ortega C, Trexler M, Tague N, Law B, Benman WKJ, Letendre J, Beal J, Wong WW (2019) High-performance chemical- and light-inducible recombinases in mammalian cells and mice. Nat Commun 10(1):4845. https://doi.org/10.1038/s41467-019-12800-7

    Article  CAS  Google Scholar 

  22. Bessen JL, Afeyan LK, Dančík V, Koblan LW, Thompson DB, Leichner C, Clemons PA, Liu DR (2019) High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases. Nat Commun 10(1):1937. https://doi.org/10.1038/s41467-019-09987-0

    Article  CAS  Google Scholar 

  23. Liu W, Tuck LR, Wright JM, Cai Y (2017) Using purified tyrosine site-specific recombinases in vitro to rapidly construct and diversify metabolic pathways. Methods Mol Biol (Clifton, NJ) 1642:285–302. https://doi.org/10.1007/978-1-4939-7169-5_18

    Article  CAS  Google Scholar 

  24. Koch B, Nijmeijer B, Kueblbeck M, Cai Y, Walther N, Ellenberg J (2018) Generation and validation of homozygous fluorescent knock-in cells using CRISPR-Cas9 genome editing. Nat Protoc 13(6):1465–1487. https://doi.org/10.1038/nprot.2018.042

    Article  CAS  Google Scholar 

  25. Sakuma T, Nakade S, Sakane Y, Suzuki KT, Yamamoto T (2016) MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nat Protoc 11(1):118–133. https://doi.org/10.1038/nprot.2015.140

    Article  CAS  Google Scholar 

  26. Shimshek DR, Kim J, Hübner MR, Spergel DJ, Buchholz F, Casanova E, Stewart AF, Seeburg PH, Sprengel R (2002) Codon-improved Cre recombinase (iCre) expression in the mouse. Genesis (New York, NY: 2000) 32(1):19–26. https://doi.org/10.1002/gene.10023

    Article  CAS  Google Scholar 

  27. Sauer B, McDermott J (2004) DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages. Nucleic Acids Res 32(20):6086–6095. https://doi.org/10.1093/nar/gkh941

    Article  CAS  Google Scholar 

  28. Anastassiadis K, Fu J, Patsch C, Hu S, Weidlich S, Duerschke K, Buchholz F, Edenhofer F, Stewart AF (2009) Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice. Dis Model Mech 2(9–10):508–515. https://doi.org/10.1242/dmm.003087

    Article  CAS  Google Scholar 

  29. Madisen L, Garner AR, Shimaoka D, Chuong AS, Klapoetke NC, Li L, van der Bourg A, Niino Y, Egolf L, Monetti C, Gu H, Mills M, Cheng A, Tasic B, Nguyen TN, Sunkin SM, Benucci A, Nagy A, Miyawaki A, Helmchen F, Empson RM, Knöpfel T, Boyden ES, Reid RC, Carandini M, Zeng H (2015) Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85(5):942–958. https://doi.org/10.1016/j.neuron.2015.02.022

    Article  CAS  Google Scholar 

  30. Karimova M, Abi-Ghanem J, Berger N, Surendranath V, Pisabarro MT, Buchholz F (2013) Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system. Nucleic Acids Res 41(2):e37. https://doi.org/10.1093/nar/gks1037

    Article  CAS  Google Scholar 

  31. Karimova M, Splith V, Karpinski J, Pisabarro MT, Buchholz F (2016) Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering. Sci Rep 6:30130. https://doi.org/10.1038/srep30130

    Article  CAS  Google Scholar 

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Acknowledgments

This research was supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED under Grant Number JP21am0101119 and JSPS KAKENHI Grant Number JP21K06131.

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Authors and Affiliations

  1. Department of Frontier Research and Development, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan

    Manabu Nakayama

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  1. Manabu Nakayama
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Correspondence to Manabu Nakayama .

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Editors and Affiliations

  1. Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan

    Izuho Hatada

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Nakayama, M. (2023). VCre/VloxP and SCre/SloxP as Reliable Site-Specific Recombination Systems for Genome Engineering. In: Hatada, I. (eds) Genome Editing in Animals. Methods in Molecular Biology, vol 2637. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3016-7_13

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  • DOI: https://doi.org/10.1007/978-1-0716-3016-7_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3015-0

  • Online ISBN: 978-1-0716-3016-7

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