July 19, 2019  |  

Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing.

Authors: Hendel, Ayal and Kildebeck, Eric J and Fine, Eli J and Clark, Joseph T and Punjya, Niraj and Sebastiano, Vittorio and Bao, Gang and Porteus, Matthew H

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 novel method for quantifying individual genome editing outcomes at any site of interest using single molecule real time (SMRT) DNA sequencing. We show that this approach can be applied at various loci, using multiple engineered nuclease platforms including TALENs, RNA guided endonucleases (CRISPR/Cas9), and ZFNs, and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach facilitates the evaluation of new gene editing technologies and permits sensitive quantification of editing outcomes in almost every experimental system used.

Journal: Cell reports
DOI: 10.1016/j.celrep.2014.02.040
Year: 2014

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