Toward Precision Molecular Surgery: Robust, Selective Induction of Microhomology-mediated End Joining in vivo

Abstract

One key problem in precision genome editing is the resultant unpredictable plurality of sequence outcomes at the site of targeted DNA double-strand 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 identical alleles using NHEJ is a labor intensive process. In this study, we propose inducing 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 as a training dataset, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation and prospectively designed MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated a high level of mutant allele homogeneity at these loci. F0 mutant zebrafish embryos and larvae generated with these gRNAs faithfully recapitulated previously reported, recessive loss-of-function phenotypes. We also provide a novel algorithm MENTHU (http://genesculpt.org/menthu/) for improved prediction of candidate MMEJ loci, suitable for both targeted and genome-wide applications. We believe that this MMEJ-centric approach will have a broad impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of a 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.\n\nAuthor SummaryNew gene editing tools precisely break DNA at pre-defined genomic locations, but cells repair these lesions using diverse pathways that often lead to unpredictable outcomes in the resulting DNA sequences. This sequence diversity in gene editing outcomes represents an important obstacle to the application of this technology for human therapies. Using a vertebrate animal as a model system, we provide strong evidence that we can overcome this obstacle by selectively directing DNA repair of double-stranded breaks through a lesser-described pathway termed Microhomology-mediated End Joining (MMEJ). Unlike other, better-understood pathways, MMEJ uses recurring short sequence patterns surrounding the site of DNA breakage. This enables the prediction of repair outcomes with improved accuracy. Importantly, we also show that preferential activation of MMEJ is compatible with effective gene editing. Finally, we provide a simple algorithm and software for designing DNA-breaking reagents that have high chance of activating the MMEJ pathway. We believe that the MMEJ-centric approach to be broadly applicable for a variety of gene editing applications both within the laboratory and for human therapies.\n\nAuthor ContributionHA contributed in Conceptualization, Data Curation, Formal Analysis, Investigation, Funding Acquisition, Methodology, Validation, Visualization, Writing - Original draft preparation, and Writing - Review and Editing. TLE contributed in Data Curation, Investigation, Writing - Original draft preparation, and Writing - Review and Editing. GMG contributed in Software, Validation, and Writing. CMM contributed in Software Validation, and Writing. AVD contributed in Investigation, Methodology, Validation, and Writing - Review and Editing. KJS contributed in Investigation and Writing - Review and Editing. ACM contributed in Conceptualization, Data Curation, Investigation, and Writing - Review and Editing. DD contributed in Funding Acquisition, Resources, and Writing - Review and Editing. KJC contributed in Conceptualization, Funding Acquisition, Resources, Supervision, and Writing - Review and Editing. SCE contributed in Conceptualization, Funding Acquisition, Project Administration, Resources, Supervision, Writing - Review and Editing.