Journal
CELL REPORTS
Volume 7, Issue 1, Pages 293-305Publisher
CELL PRESS
DOI: 10.1016/j.celrep.2014.02.040
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Funding
- National Institutes of Health as an NIH Nanomedicine Development Center Award [PN2EY018244]
- NIH from the NIAID [R01 AI097320]
- Myotonic Dystrophy Foundation (MDF)
- Dean's postdoctoral fellowship award at the Stanford School of Medicine
- NSF Graduate Research Fellowship [DGE-1148903]
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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.
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