title: A T7 Endonuclease I Assay to Detect Talen-Mediated Targeted Mutation of HBV cccDNA.
abstract: Gene editing using designer nucleases is now widely used in many fields of molecular biology. The technology is being developed for the treatment of viral infections such as persistant hepatitis B virus (HBV). The replication intermediate of HBV comprising covalently closed circular DNA (cccDNA) is stable and resistant to available licensed antiviral agents. Advancing gene editing as a means of introducing targeted mutations into cccDNA thus potentially offers the means to cure infection by the virus. Essentially, targeted mutations are initiated by intracellular DNA cleavage, then error-prone nonhomologous end joining results in insertions and deletions (indels) at intended sites. Characterization of these mutations is crucial to confirm activity of potentially therapeutic nucleases. A convenient tool for evaluation of the efficiency of target cleavage is the single strand-specific endonuclease, T7EI. Assays employing this enzyme entail initial amplification of DNA encompassing the targeted region. Thereafter the amplicons are denatured and reannealed to allow hybridization between indel-containing and wild-type sequences. Heteroduplexes that contain mismatched regions are susceptible to action by T7EI and cleavage of the hybrid amplicons may be used as an indicator of efficiency of designer nucleases. The protocol described here provides a method of isolating cccDNA from transfected HepG2.2.15 cells and evaluation of the efficiency of mutation by a transcription activator-like effector nuclease that targets the surface open reading frame of HBV.
authors.affiliation: Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Health Sciences Faculty, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa.|University Medical Center Freiburg, Institute for Cell and Gene Therapy & Center for Chronic Immunodeficiency, Breisacherstrasse 115, 79106, Freiburg, Germany.
authors.forename: Kristie
authors.initials: K
authors.lastname: Bloom
authors.affiliation: Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Health Sciences Faculty, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa.
authors.forename: Abdullah
authors.initials: A
authors.lastname: Ely
authors.affiliation: Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Health Sciences Faculty, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa. Patrick.Arbuthnot@wits.ac.za.
authors.forename: Patrick
authors.initials: P
authors.lastname: Arbuthnot
mesh_terms: D004270:DNA, Circular; D004279:DNA, Viral; D003850:Deoxyribonuclease I / Q000378:metabolism*; D015870:Gene Expression; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D054643:INDEL Mutation; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D000069896:Transcription Activator-Like Effector Nucleases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral; D000069896:Transcription Activator-Like Effector Nucleases; D003850:Deoxyribonuclease I
keywords: HBV; Indels; T7EI; TALEN; cccDNA