A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals: A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis

Vanessa Chenouard; Lucas Brusselle; Jean-Marie Heslan; Séverine Remy; Séverine Ménoret; Claire Usal; Laure-Hélène Ouisse; Tuan Huy NGuyen; Ignacio Anegon; Laurent Tesson

doi:10.1016/j.jgg.2016.04.005

Volume 43 Issue 5

May 2016

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Article Navigation > Journal of Genetics and Genomics > 2016 > 43(5): 341-348

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A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals: A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis

doi: 10.1016/j.jgg.2016.04.005

a
Institut National de la Santé Et de la Recherche Médicale UMR 1064-Institut Transplantation Urologie-Néphrologie, Centre Hospitalier Universitaire de Nantes, Nantes 44093, France
b
Transgenesis Rat ImmunoPhenomic Platform, Structure Fédérative de Recherche François Bonamy, Centre National de Recherche Scientifique UMS3556, Nantes 44093, France
c
GenoCellEdit, INSERM UMR 1064-ITUN, CHU Nantes, Nantes 44093, France

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Corresponding author: E-mail address: laurent.tesson@univ-nantes.fr (Laurent Tesson)
Received Date: 2016-01-15
Accepted Date: 2016-04-05
Rev Recd Date: 2016-03-30

Available Online: 2016-05-04

Publish Date: 2016-05-20

Abstract

Abstract

The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species. Moreover, these novel tools have become easier to use and have resulted in a great increase of applications. Whilst gene knockout (KO) or knockin (KI) animal models are relatively easy to achieve, there is a bottleneck in the detection and analysis of these mutations. Although several methods exist to detect these targeted mutations, we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process. The HMA-CE method uses a simple PCR amplification of genomic DNA (gDNA) followed by an automated capillary electrophoresis step which reveals a heteroduplexes (HD) signature for each mutation. This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.
- Genome editing,
- Genotyping,
- Heteroduplex mobility assay,
- Capillary electrophoresis

These two authors contributed equally to this work.

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References(17)

References

[1]	Ansai, S., Inohaya, K., Yoshiura, Y. et al. Design, evaluation, and screening methods for efficient targeted mutagenesis with transcription activator-like effector nucleases in medaka Dev. Growth Differ., 56 (2014),pp. 98-107
[2]	Chandrasegaran, S., Carroll, D. Origins of programmable nucleases for genome engineering J. Mol. Biol., 428 (2015),pp. 963-989
[3]	Chen, J., Zhang, X., Wang, T. et al. Efficient detection, quantification and enrichment of subtle allelic alterations DNA Res., 19 (2012),pp. 423-433
[4]	Crispo, M., Mulet, A.P., Tesson, L. et al. Efficient generation of myostatin knock-out sheep using CRISPR/Cas9 technology and microinjection into zygotes PLoS One, 10 (2015),p. e0136690
[5]	Judo, M.S., Wedel, A.B., Wilson, C. Stimulation and suppression of PCR-mediated recombination Nucleic Acids Res., 26 (1998),pp. 1819-1825
[6]	Larcher, T., Lafoux, A., Tesson, L. et al. Characterization of dystrophin deficient rats: a new model for Duchenne muscular dystrophy PLoS One, 9 (2014),p. e110371
[7]	Menoret, S., De Cian, A., Tesson, L. et al. Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins Sci. Rep., 5 (2015),p. 14410
[8]	Menoret, S., Fontaniere, S., Jantz, D. et al. FASEB J., 27 (2013),pp. 703-711
[9]	Ota, S., Hisano, Y., Muraki, M. et al. Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays Genes Cells, 18 (2013),pp. 450-458
[10]	Ranchoux, B., Antigny, F., Rucker-Martin, C. et al. Endothelial-to-mesenchymal transition in pulmonary hypertension Circulation, 131 (2015),pp. 1006-1018
[11]	Remy, S., Tesson, L., Menoret, S. et al. Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases Genome Res., 24 (2014),pp. 1371-1383
[12]	Tan, W., Proudfoot, C., Lillico, S.G. et al. Gene targeting, genome editing: from Dolly to editors Transgenic Res. (2016)
[13]	Thomas, H.R., Percival, S.M., Yoder, B.K. et al. High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis PLoS One, 9 (2014),p. e114632
[14]	Thomas, K.R., Capecchi, M.R. Nature, 346 (1990),pp. 847-850
[15]	Vouillot, L., Thelie, A., Pollet, N. Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases G3 (Bethesda), 5 (2015),pp. 407-415
[16]	Yang, Z., Steentoft, C., Hauge, C. et al. Fast and sensitive detection of indels induced by precise gene targeting Nucleic Acids Res., 43 (2015),p. e59
[17]	Zhu, X., Xu, Y., Yu, S. et al. An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system Sci. Rep., 4 (2014),p. 6420