Please use this identifier to cite or link to this item: http://hdl.handle.net/10397/80294
Title: Robust activation of microhomology-mediated end joining for precision gene editing applications
Authors: Ata, H
Ekstrom, TL
Martinez-Galvez, G
Mann, CM
Dvornikov, AV
Schaefbauer, KJ
Ma, AC 
Dobbs, D
Clark, KJ
Ekker, SC
Issue Date: 2018
Publisher: Public Library of Science
Source: PLoS genetics, Sept. 2018, v. 14, no. 9, e1007652, p. 1-22 How to cite?
Journal: PLoS genetics 
Abstract: One key problem in precision genome editing is the unpredictable plurality of sequence outcomes at the site of targeted DNA double stranded breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of the identical alleles using NHEJ is a labor intensive process. In this study, we propose Microhomology-mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% similar to 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation to prospectively design MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated high levels of mutant allele homogeneity. MMEJ-based DNA repair at these target loci successfully generated FO mutant zebrafish embryos and larvae that faithfully recapitulated previously reported, recessive, loss-of-function phenotypes. We also tested the generalizability of our approach in cultured human cells. Finally, we provide a novel algorithm, MENTHU (http://genesculpt.org/menthu/), for improved and facile prediction of candidate MMEJ loci. We believe that this MMEJ-centric approach will have a broader impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an endogenous target will have important implications for gene therapy applications in human genetic diseases.
URI: http://hdl.handle.net/10397/80294
ISSN: 1553-7390
EISSN: 1553-7404
DOI: 10.1371/journal.pgen.1007652
Rights: © 2018 Ata et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The following publication Ata, H., Ekstrom, T.L., Martinez-Galvez, G., Mann, C.M., Dvornikov, A.V., Schaefbauer, K.J., ... & Ekker, S.C. (2018). Robust activation of microhomology-mediated end joining for precision gene editing applications. PLoS genetics, 14 (9), e1007652, p. 1-22 is available at https://dx.doi.org/10.1371/journal.pgen.1007652
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