Sensitive and Quantitative Measurement of Nuclease Mediated Genome Editing at Human Endogenous Loci using SMRT Sequencing. A. Hendel1, E. Kildebeck1, E. Fine2, J. Clark1, G. Bao2, M. Porteus1 1) Department of Pediatrics, Stanford University, Stanford, CA; 2) Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta.

   Targeted genome editing with engineered nucleases allows researchers to introduce precise sequence modifications at almost any site within the genome. These genome modification strategies are initiated by a double-strand break created by an engineered nuclease (e.g. zinc finger nuclease, TAL effector nuclease, or an RNA-guided endonuclease). The precise genome modification, however depends on whether the break is repaired in a mutagenic fashion by non-homologous end-joining (NHEJ) or using a provided donor DNA molecule by homologous recombination (HR). If the break is repaired by NHEJ, small insertions or deletions are created at the site of the break. If the break is repaired by HR, however, defined sequence changes, can be introduced at the site of the break creating nucleotide specific modifications to the genome. An important aspect is to develop sensitive and quantitative methods to measure these two different outcomes at human endogenous loci. While there are different reporter based assays to measure these two events, our work is the first that allows simultaneous measurement of both events at endogenous loci without having to use reporter genes. Because of the large size of donor DNA repair templates used in genome editing, there has not been a sequencing platform capable of providing a comprehensive picture of how frequently NHEJ is occurring alongside targeted HR. We now show that the long read lengths afforded by Single Molecule, Real-Time (SMRT) sequencing provide the platform needed to simultaneously measure genome editing outcomes by either mutagenic NHEJ or by HR at endogenous human loci. Using this method, we were able to analyze the frequency of multiple genome editing outcomes simultaneously at an endogenous locus in human cell lines with a detection sensitivity of ~ 0.1%. We used this method to investigate the variables critical to increasing targeted genome editing, determine conditions that maximize HR while minimizing mutagenic NHEJ, and measure genome editing in difficult to target hematopoietic stem and progenitor cells. In sum, our new method to quantify genome editing outcomes has significant advantage over prior methods, because it does not require making a stable reporter cell line, it measures outcomes at endogenous loci, it is sensitive to an unprecedented level, and it can be utilized in cell types for which creating reporter cell lines is simply not possible including human hematopoietic stem and progenitor cells.

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