Digital detection of endonuclease mediated gene disruption in the HIV provirus

Scientific Reports
Ruth Hall SedlakKeith R Jerome

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.

References

Jul 1, 1977·The Journal of General Virology·F L GrahamR Nairn
Apr 20, 2004·BioTechniques·Peter QiuGary F Gerard
Oct 6, 2004·Genome Biology·Robert C GentlemanJianhua Zhang
Dec 5, 2006·PLoS Pathogens·Joshua L AndersenVicente Planelles
Jan 28, 2009·Marine Drugs·Cynthia D AndjelicLouis R Barrows
May 24, 2011·Methods in Enzymology·Daniel G Gibson
Nov 30, 2011·Analytical Chemistry·Leonardo B PinheiroKerry R Emslie
Mar 6, 2012·Nature Methods·Ben Langmead, Steven L Salzberg
Jun 22, 2012·Journal of Virology·Joshua T SchifferKeith R Jerome
Sep 18, 2012·Current Opinion in Immunology·Stacey E Wirt, Matthew H Porteus
Nov 28, 2012·Diagnostic Microbiology and Infectious Disease·Ruth Hall Sedlak, Keith R Jerome
Feb 21, 2013·Methods in Molecular Biology·Sarah K BaxterJordan Jarjour
Mar 13, 2013·Current Opinion in HIV and AIDS·Daniel StoneKeith R Jerome
May 15, 2013·Trends in Biotechnology·Thomas GajCarlos F Barbas
Nov 29, 2013·Nucleic Acids Research·Sandrine BoisselAndrew M Scharenberg
Feb 6, 2014·Molecular Therapy. Nucleic Acids·Martine AubertKeith R Jerome
Apr 4, 2014·Bioinformatics·Anthony M BolgerBjoern Usadel
Oct 11, 2014·Nucleic Acids Research·Eva K BrinkmanBas van Steensel
Nov 20, 2014·Methods in Molecular Biology·Sandrine Boissel, Andrew M Scharenberg
Jan 9, 2015·G3 : Genes - Genomes - Genetics·Léna VouillotNicolas Pollet
Feb 6, 2015·Nature Medicine·David Benjamin Turitz CoxFeng Zhang
May 9, 2015·The Journal of Molecular Diagnostics : JMD·Pierre-Jean LamyAnne-Claire Laberenne

❮ Previous
Next ❯

Citations

Apr 20, 2016·The Journal of Antimicrobial Chemotherapy·Pavitra RoychoudhuryJoshua T Schiffer
Nov 18, 2016·AIDS Patient Care and STDs·Keith R Jerome
May 25, 2016·Genes to Cells : Devoted to Molecular & Cellular Mechanisms·Mitsuki ShigetaKen-Ichi T Suzuki
Jun 15, 2018·Expert Review of Molecular Diagnostics·Alexander L Greninger
Mar 17, 2017·Journal of Clinical Microbiology·Jane Kuypers, Keith R Jerome
Apr 1, 2016·Current Opinion in HIV and AIDS·Chelsea SpraggKeith R Jerome
Feb 18, 2017·Cellular and Molecular Life Sciences : CMLS·Kamel KhaliliJeffrey M Jacobson
Sep 20, 2016·JCI Insight·Martine AubertKeith R Jerome
Sep 19, 2017·Analytical and Bioanalytical Chemistry·Alexandra Bogožalec KoširJana Žel
Feb 5, 2021·Journal of Medical Virology·Amir Asri KojabadMajid Mirzaei Nodooshan

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
transfection
Illumina sequencing
flow cytometry
targeted mutation
cleavage assay

Software Mentioned

Trimmomatic
TIDE
Galaxy
Rsamtools
ShortRead
custom
Cutadapt
Biostrings
Primer Express
Bioconductor

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.