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.
|Number of pages||13|
|State||Published - 4 Oct 2014|
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health as an NIH Nanomedicine Development Center Award (PN2EY018244 to M.H.P. and G.B.). This work was also supported by NIH grant R01 AI097320 from the NIAID to M.H.P. M.H.P. also thanks the Laurie Krauss Lacob Faculty Scholar Award for ongoing support. A.H. was supported in part by the Fund-A-Fellow postdoctoral fellowship research grant award from the Myotonic Dystrophy Foundation (MDF) and the Dean’s postdoctoral fellowship award at the Stanford School of Medicine. E.J.F. was supported by the NSF Graduate Research Fellowship under Grant No. DGE-1148903. We thank Dr. Norma Neff, Ben Passarelli, Jad Kanbar, and Pacific Biosciences for help with the SMRT sequencing technology. We thank Prof. Stephen Quake, Prof. Michael Snyder, and Prof. Joseph Puglisi for letting us using their PacBio RS instruments. We thank Shondra Miller, John Eid, Jonas Korlach, Aaron Straight, and Porteus and Bao lab members for helpful comments and discussion.