Biomedical Science Letters (대한의생명과학회지)

The Korean Society for Biomedical Laboratory Sciences (대한의생명과학회)
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Efficient Production of loxP Knock-in Mouse using CRISPR/Cas9 System

  • Jung, Sundo (Department of Biomedical Laboratory Science, Shinhan University)
  • Received : 2020.05.14
  • Accepted : 2020.06.16
  • Published : 2020.06.30
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Abstract

Of the various types of mice used for genome editing, conditional knock-out (cKO) mice serve as an important model for studying the function of genes. cKO mice can be produced using loxP knock-in (KI) mice in which loxP sequences (34 bp) are inserted on both sides of a specific region in the target gene. These mice can be used as KO mice that do not express a gene at a desired time or under a desired condition by cross-breeding with various Cre Tg mice. Genome editing has been recently made easy by the use of third-generation gene scissors, the CRISPR-Cas9 system. However, very few laboratories can produce mice for genome editing. Here we present a more efficient method for producing loxP KI mice. This method involves the use of an HDR vector as the target vector and ssODN as the donor DNA in order to induce homologous recombination for producing loxP KI mice. On injecting 20 ng/µL of ssODN, it was observed that the target exon was deleted or loxP was inserted on only one side. However, on injecting 10 ng/µL of the target HDR vector, the insertion of loxP was observed on both sides of the target region. In the first PCR, seven mice were identified to be loxP KI mice. The accuracy of their gene sequences was confirmed through Sanger sequencing. It is expected that the loxP KI mice produced in this study will serve as an important tool for identifying the function of the target gene.

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References

  1. Channabasavaiah B, Gurumurthy BC, Sato M, Nakamura A, Inui M, Kawano N, Islam AM, Ogiwara S, Takabayshi S, Matsuyama M, Nakagawa S, Miura H, Ohtsuka M. Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD. Nat Prot. 2019. 14: 2452-2482. https://doi.org/10.1038/s41596-019-0187-x
  2. Cho SW, Kim S, Kim JM, Kim JS. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013. 31: 230-232. https://doi.org/10.1038/nbt.2507
  3. Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kuhn R. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. 2015. 33: 543-548. https://doi.org/10.1038/nbt.3198
  4. Chu VT, Weber T, Graf R, Sommermann T, Petsch K, Sack U, Volchkov P, Rajewsky K, Kuhn R. Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes. BMC Biotechnol. 2016. 16: 86-97. https://doi.org/10.1186/s12896-016-0316-3
  5. Codner FG, Mianne J, Caulder A, Loeffler J, Fell R, King R, Allan A, Mackenzie M, Pike JF, McCabe VC, et al. Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants. BMC Biol. 2018. 16: 70. https://doi.org/10.1186/s12915-018-0530-7
  6. Cox DBT, Platt JR, Zhang F. Therapeutic Genome Editing: Prospects and Challenges. Nat Med. 2015. 21: 121-131. https://doi.org/10.1038/nm.3793
  7. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013. 31: 822-826. https://doi.org/10.1038/nbt.2623
  8. Gurumurthy BC, O'Brien RA, Quadros MR, Adams J, Alcaide P, Ayabe S, Ballard J, Batra KS, Beauchamp MC, Becker AK, et al. Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation. Genome Biol. 2019. 20: 171-184. https://doi.org/10.1186/s13059-019-1776-2
  9. Horii T, Arai Y, Yamazaki M, Morita S, Kimura M, Itoh M, Abe Y, Hatada I. Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering. Sci Rep. 2014. 4: 4513-4518. https://doi.org/10.1038/srep04513
  10. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang, F. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013. 31: 827-832. https://doi.org/10.1038/nbt.2647
  11. Ittner LM, Gotz J. Pronuclear injection for the production of transgenic mice. Nat Protoc. 2007. 2: 1206-1215. https://doi.org/10.1038/nprot.2007.145
  12. Jung S. Efficient generation of human IgG1 light kappa constant region knock-in mouse by CRISPR/Cas9 system. Biomedical Science Letters. 2019. 25: 372-380. https://doi.org/10.15616/BSL.2019.25.4.372
  13. Lanza GD, Gaspero A, Lorenzo I, Liao L, Zheng P, Wang Y, Deng Y, Cheng C, Zhang C, Seavitt RJ, DeMayo FJ, Xu J, Dickinson ME, Beaudet AL, Heaney JD. Comparative analysis of singlestranded DNA donors to generate conditional null mouse alleles. BMC Biology. 2018. 16: 69. https://doi.org/10.1186/s12915-018-0529-0
  14. Li Q, Qin Z, Wang Q, Xu T, Yang Y, He Z. Applications of genome editing technology in animal disease modeling and gene therapy. Computational and Structural Biotechnology Journal. 2019. 17: 689-698. https://doi.org/10.1016/j.csbj.2019.05.006
  15. Ma X, Chen C, Veevers J, Zhou X, Ross RS, Feng W, Chen J. CRISPR/Cas9-mediated gene manipulation to create singleamino-acid-substituted and foxed mice with a cloning-free method. Sci Rep. 2017. 7: 42244-42252. https://doi.org/10.1038/srep42244
  16. Okamoto S, Amnaishi Y, Maki I, Enoki T, Mineno J. Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs. Sci Rep. 2019. 9: 4811-4821. https://doi.org/10.1038/s41598-019-41121-4
  17. Raveux A, Vandormael-Pournin S, Cohen-Tannoudji M. Optimization of the production of knock-in alleles by CRISPR/Cas9 microinjection into the mouse zygote. Sci Rep. 2017. 7: 42661-42670. https://doi.org/10.1038/srep42661
  18. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013. 145: 1380-1389.
  19. Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Nactl Acad Sci. 1988. 85: 5166-5170. https://doi.org/10.1073/pnas.85.14.5166
  20. Skarnes WC, Rosen B, West AP, Koutsourakis M, Bushell W, Iyer V, Mujica AO, Thomas M, Harrow J, Cox T, Jackson D, Severin J, Biggs P, Fu J, Nefedov M, de Jong PJ, Stewart AF, Bradley A. A conditional knockout resource for the genomewide study of mouse gene function. Nature. 2011. 474: 337-342. https://doi.org/10.1038/nature10163
  21. Yan H, Wang H, Jaenisch R. Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. Nat Protoc. 2014. 9: 1956-1968. https://doi.org/10.1038/nprot.2014.134
  22. Yu Y, Bredley A. Engineering chromosomal rearrangements in mice. Nat Rev Genet. 2001. 2: 780-790. https://doi.org/10.1038/35093564