title: Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing.
abstract: Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a method for quantifying individual genome-editing outcomes at any site of interest with single-molecule real-time (SMRT) DNA sequencing. We show that this approach can be applied at various loci using multiple engineered nuclease platforms, including transcription-activator-like effector nucleases (TALENs), RNA-guided endonucleases (CRISPR/Cas9), and zinc finger nucleases (ZFNs), and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach offers a technique for studying double-strand break repair, facilitates the evaluation of gene-editing technologies, and permits sensitive quantification of editing outcomes in almost every experimental system used.
authors.affiliation: Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
authors.forename: Ayal
authors.initials: A
authors.lastname: Hendel
authors.affiliation: Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Kildebeck
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
authors.forename: Eli J
authors.initials: EJ
authors.lastname: Fine
authors.affiliation: Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Clark
authors.affiliation: Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
authors.forename: Niraj
authors.initials: N
authors.lastname: Punjya
authors.affiliation: Department of Obstetrics and Gynecology, Stanford University, Stanford, CA 94305, USA.
authors.forename: Vittorio
authors.initials: V
authors.lastname: Sebastiano
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Bao
authors.affiliation: Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Porteus
mesh_terms: D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004720:Endonucleases / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D015894:Genome, Human; D006801:Humans; D020014:K562 Cells; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA / Q000379:methods*; D014162:Transfection
chemical_list: D004720:Endonucleases