METHOD

Modification of the method for analysis of genome editing results using CRISPR/Cas9 system on preimplantation mouse embryos

About authors

1 Marlin Biotech, Moscow, Russia

2 Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia

3 Department of Neurology, Neurosurgery and Medical Genetics, Medical Faculty,
Pirogov Russian National Research Medical University, Moscow, Russia

Correspondence should be addressed: Tatiana Dimitrieva
ul. Vavilova, d. 34/5, Moscow, Russia, 119334; moc.liamg@at.aveirtimid

About paper

Acknowledgements: authors thank the Shared Resource Center of the Institute of Gene Biology of Russian Academy of Sciences for the equipment provided for this research.

Received: 2016-06-16 Accepted: 2016-06-21 Published online: 2017-01-05
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Genetically modified animals are an important tool for biomedical research. The CRISPR/Cas9 editing genome system is increasingly being used for production of such animals. Through microinjection, complex with guide RNA and Cas9 protein is delivered in fertilized eggs from which the animal subsequently develops with a modification in the genome. Generally, analysis of the specificity and efficiency of the system in each case is carried out after obtaining a progeny with the likely mutation. However, analysis at the preimplantation stage would allow reducing the time of the experiment, as well as understanding the reason for the birth of a small number of transgenic animals, or even lack of them in the offsprings. The paper proposes a modification of the method of preparation of total DNA from mouse blastocysts. The modification allows to easier and faster detect the results of microinjection of the CRISPR/Cas9 complex in the zygote. Having applied the method described in this paper, we successfully identified short deletions in intron 34 of dystrophin gene (DMD) in 12 out of 13 treated embryos and insertion in the break site in intron 8 of the DMD gene in 11 out of 21 samples analyzed. Using for analysis the total DNA prepared by the method proposed, you can analyze up to 20 different sites in the mouse embryo genome at the blastocyst stage without the need for full genomic amplification.

Keywords: genome editing, CRISPR/Cas9, short nucleotide insertions, nucleotide deletions, mouse embryos, Duchenne muscular dystrophy

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