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Digital detection of endonuclease mediated gene disruption in the HIV provirus

Genome editing by designer nucleases is a rapidly evolving technology utilized in a highly diverse set of research fields. Among all fields, the T7 endonuclease mismatch cleavage assay, or Surveyor assay, is the most commonly used tool to assess genomic editing by designer nucleases. This assay, whi...

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Detalles Bibliográficos
Autores principales: Sedlak, Ruth Hall, Liang, Shu, Niyonzima, Nixon, De Silva Feelixge, Harshana S., Roychoudhury, Pavitra, Greninger, Alexander L., Weber, Nicholas D., Boissel, Sandrine, Scharenberg, Andrew M., Cheng, Anqi, Magaret, Amalia, Bumgarner, Roger, Stone, Daniel, Jerome, Keith R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4735761/
https://www.ncbi.nlm.nih.gov/pubmed/26829887
http://dx.doi.org/10.1038/srep20064
Descripción
Sumario:Genome editing by designer nucleases is a rapidly evolving technology utilized in a highly diverse set of research fields. Among all fields, the T7 endonuclease mismatch cleavage assay, or Surveyor assay, is the most commonly used tool to assess genomic editing by designer nucleases. This assay, while relatively easy to perform, provides only a semi-quantitative measure of mutation efficiency that lacks sensitivity and accuracy. We demonstrate a simple droplet digital PCR assay that quickly quantitates a range of indel mutations with detection as low as 0.02% mutant in a wild type background and precision (≤6%CV) and accuracy superior to either mismatch cleavage assay or clonal sequencing when compared to next-generation sequencing. The precision and simplicity of this assay will facilitate comparison of gene editing approaches and their optimization, accelerating progress in this rapidly-moving field.