title: Digital detection of endonuclease mediated gene disruption in the HIV provirus.
abstract: 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.
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Ruth Hall
authors.initials: RH
authors.lastname: Sedlak
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Shu
authors.initials: S
authors.lastname: Liang
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98102, USA.
authors.forename: Nixon
authors.initials: N
authors.lastname: Niyonzima
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98102, USA.
authors.forename: Harshana S
authors.initials: HS
authors.lastname: De Silva Feelixge
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98102, USA.
authors.forename: Pavitra
authors.initials: P
authors.lastname: Roychoudhury
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Alexander L
authors.initials: AL
authors.lastname: Greninger
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98102, USA.
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Weber
authors.affiliation: Seattle Children's Research Institute, Seattle, WA, 98101, USA.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Boissel
authors.affiliation: Seattle Children's Research Institute, Seattle, WA, 98101, USA.|University of Washington School of Medicine, Seattle, WA, 98195, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scharenberg
authors.affiliation: Department of Biostatistics, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Anqi
authors.initials: A
authors.lastname: Cheng
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, WA, 98195, USA.|Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98102, USA.|Department of Biostatistics, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Amalia
authors.initials: A
authors.lastname: Magaret
authors.affiliation: Department of Microbiology, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Roger
authors.initials: R
authors.lastname: Bumgarner
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98102, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Stone
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, WA, 98195, USA.|Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98102, USA.
authors.forename: Keith R
authors.initials: KR
authors.lastname: Jerome
mesh_terms: D005090:Exodeoxyribonucleases / Q000737:chemistry*; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D054643:INDEL Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics*
chemical_list: D005090:Exodeoxyribonucleases; C046264:gene b exonuclease
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