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Germini D, Bou Saada Y, Tsfasman T, et al. A One-Step PCR-Based Assay to Evaluate the Efficiency and Precision of Genomic DNA-Editing Tools. Mol Ther Methods Clin Dev. 2017;5:43-50doi: 10.1016/j.omtm.2017.03.001.
Germini, D., Bou Saada, Y., Tsfasman, T., Osina, K., Robin, C., Lomov, N., Rubtsov, M., Sjakste, N., Lipinski, M., & Vassetzky, Y. (2017). A One-Step PCR-Based Assay to Evaluate the Efficiency and Precision of Genomic DNA-Editing Tools. Molecular therapy. Methods & clinical development, 543-50. https://doi.org/10.1016/j.omtm.2017.03.001
Germini, Diego, et al. "A One-Step PCR-Based Assay to Evaluate the Efficiency and Precision of Genomic DNA-Editing Tools." Molecular therapy. Methods & clinical development vol. 5 (2017): 43-50. doi: https://doi.org/10.1016/j.omtm.2017.03.001
Germini D, Bou Saada Y, Tsfasman T, Osina K, Robin C, Lomov N, Rubtsov M, Sjakste N, Lipinski M, Vassetzky Y. A One-Step PCR-Based Assay to Evaluate the Efficiency and Precision of Genomic DNA-Editing Tools. Mol Ther Methods Clin Dev. 2017 Mar 08;5:43-50. doi: 10.1016/j.omtm.2017.03.001. eCollection 2017 Jun 16. PMID: 28480303; PMCID: PMC5415314.
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Mol Ther Methods Clin Dev. 2017 Mar 08;5:43-50. doi: 10.1016/j.omtm.2017.03.001. eCollection 2017 Jun 16.

A One-Step PCR-Based Assay to Evaluate the Efficiency and Precision of Genomic DNA-Editing Tools.

Molecular therapy. Methods & clinical development

Diego Germini, Yara Bou Saada, Tatiana Tsfasman, Kristina Osina, Chloé Robin, Nikolay Lomov, Mikhail Rubtsov, Nikolajs Sjakste, Mar Lipinski, Yegor Vassetzky

Affiliations

  1. UMR8126, Université Paris Sud - Paris Saclay, CNRS, Institut Gustave Roussy, 94805 Villejuif, France.
  2. LIA 1066, French-Russian Joint Cancer Research Laboratory, 94805 Villejuif, France.
  3. Department of Biophysics, Institute of Physics, Nanotechnology, and Telecommunications, Peter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russia.
  4. University of Latvia, 1586 Riga, Latvia.
  5. M.V. Lomonosov Moscow State University, Moscow 119991, Russia.
  6. Department of Biochemistry and Strategic Management Department, I.M. Sechenov First Moscow State Medical University, Moscow 119048, Russia.

PMID: 28480303 PMCID: PMC5415314 DOI: 10.1016/j.omtm.2017.03.001

Abstract

Despite rapid progress, many problems and limitations persist and limit the applicability of gene-editing techniques. Making use of meganucleases, TALENs, or CRISPR/Cas9-based tools requires an initial step of pre-screening to determine the efficiency and specificity of the designed tools. This step remains time consuming and material consuming. Here we propose a simple, cheap, reliable, time-saving, and highly sensitive method to evaluate a given gene-editing tool based on its capacity to induce chromosomal translocations when combined with a reference engineered nuclease. In the proposed technique, designated engineered nuclease-induced translocations (ENIT), a plasmid coding for the DNA-editing tool to be tested is co-transfected into carefully chosen target cells along with that for an engineered nuclease of known specificity and efficiency. If the new enzyme efficiently cuts within the desired region, then specific chromosomal translocations will be generated between the two targeted genomic regions and be readily detectable by a one-step PCR or qPCR assay. The PCR product thus obtained can be directly sequenced, thereby determining the exact position of the double-strand breaks induced by the gene-editing tools. As a proof of concept, ENIT was successfully tested in different cell types and with different meganucleases, TALENs, and CRISPR/Cas9-based editing tools.

Keywords: CRISPR/Cas9; PCR; TALEN; assay; translocations

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