Quantifying Genome-Editing Outcomes at Endogenous Loci with SMRT Sequencing

• We show a method for measuring genome-editing outcomes at endogenous loci
• We show that this method is reproducible, robust, and sensitive
• The method is broadly applicable to multiple cell types and nuclease platforms
• This is a method for studying the outcomes of DNA double-strand break repair

SummaryTargeted 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.

Graphical AbstractFigure optionsDownload as PowerPoint slide