AM Bolger

B Langmead

CD Andjelic

D Stone

DB Cox

DG Gibson

EK Brinkman

FL Graham

J Goecks

JL Andersen

JT Schiffer

L Vouillot

LB Pinheiro

M Aubert

M Martin

M Morgan

MF Sanmamed

ND Weber

OS Kustikova

P Qiu

PJ Lamy

RC Gentleman

RH Sedlak

S Boissel

SE Wirt

SK Baxter

T Gaj

Scientific Reports

AbstractGenome 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.</jats:p

Ruth Hall Sedlak

Shu Liang

Nixon Niyonzima

Harshana S. De Silva Feelixge

Pavitra Roychoudhury

Alexander L. Greninger

Nicholas D. Weber

Sandrine Boissel

Andrew M. Scharenberg

Anqi Cheng

Amalia Magaret

Roger Bumgarner

Daniel Stone

Keith R. Jerome

Crossref

Digital detection of endonuclease mediated gene disruption in the HIV provirus

Digital detection of endonuclease mediated gene disruption in the HIV provirus

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