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% ~ 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 F0 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.
Bibliographical noteFunding Information:
NIH OD020166; NIH UL1TR002377; NIH GM63904; P30DK090728; NIH P30DK84567; AHA 16PRE30470004; Mayo MSTP; Mayo Foundation; and gift from Marriott Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We would like to acknowledge Melissa McNulty for TALEN synthesis and data curation, Mark Urban for zebrafish husbandry and help with microinjections, Patrick Blackburn for designing chrd #1 sgRNA, William Gendron for his assistance with human cell work, Mayo Clinic Zebrafish Facility and staff for their support, as well as Bryce Bergene and Mayo’s Creative Studio their help with figures. We also thank the Jin-Soo Kim group for developing the Microhomology-Predictor CRISPR RGEN Tool, for making source code freely available, and for sharing the deep sequencing output from their HeLa cell experiments. We thank Wesley A Wierson and Jeffrey J Essner for the tyr #2 gRNA, and the research groups of Dr. Essner and Dr Maura McGrail for valuable discussions and feedback and for MENTHU server testing, and Carolyn Lawrence-Dill and her group, especially Darwin Campbell, for valuable discussions and hosting services for MENTHU.
© 2018 Ata et al. http://creativecommons.org/licenses/by/4.0/.