title: Detecting Single-Nucleotide Substitutions Induced by Genome Editing.
abstract: The detection of genome editing is critical in evaluating genome-editing tools or conditions, but it is not an easy task to detect genome-editing events-especially single-nucleotide substitutions-without a surrogate marker. Here we introduce a procedure that significantly contributes to the advancement of genome-editing technologies. It uses droplet digital polymerase chain reaction (ddPCR) and allele-specific hydrolysis probes to detect single-nucleotide substitutions generated by genome editing (via homology-directed repair, or HDR). HDR events that introduce substitutions using donor DNA are generally infrequent, even with genome-editing tools, and the outcome is only one base pair difference in 3 billion base pairs of the human genome. This task is particularly difficult in induced pluripotent stem (iPS) cells, in which editing events can be very rare. Therefore, the technological advances described here have implications for therapeutic genome editing and experimental approaches to disease modeling with iPS cells.
authors.affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158;
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Miyaoka
authors.affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158;
authors.forename: Amanda H
authors.initials: AH
authors.lastname: Chan
authors.affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158; Departments of Medicine, and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143.
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Conklin
mesh_terms: D000818:Animals; D000072669:Gene Editing; D006801:Humans; D009711:Nucleotides / Q000032:analysis* / Q000235:genetics*; D015345:Oligonucleotide Probes / Q000235:genetics; D039904:Pluripotent Stem Cells; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D009711:Nucleotides; D015345:Oligonucleotide Probes