PROJECT SUMMARY/ABSTRACT
Nuclease-mediated homologous directed repair (HDR) in mammalian embryos remains inefficient and
unreliable despite recent advances in its development. DNA repair following cutting by nucleases manifests as
either nonhomologous end joining (NHEJ), a rapid imprecise repair mechanism, or less commonly and in the
presences of donor DNA, HDR with its perfect integration into the genome. HDR efficiency is affected by
multiple variables, including the target cell's differentiated state, donor DNA and targeting-nuclease. Current
optimization approaches using reporter constructs for HDR in preimplantation embryos do function but are
limited to active genes. Where embryos are developed to term, the testing of multiple HDR variables is
resource intense. Alternatively, using immortalized cell lines does not accurately reflect the zygote phenotype.
The broad goal of this work is to significantly improve the reliability and efficiency of targeted nuclease-
mediated HDR by developing and validating a high-throughput, low-cost approach capable of testing multiple
variables for targeting HDR directly in mouse zygotes, and making this information publicly available. In our
innovative approach, pools of ~50 zygotes are microinjected with a regent test regime (i.e., testing singly, the
multiple variables involved), these pools are grown in vitro and harvested at E3.0, and analyzed for targeting
efficiency via DNA sequencing focusing on and spanning the targeted region. This uses a single-molecule real-
time high-throughput sequencing (HTS) system yielding ~5,000, 8-10kb amplicon reads/regime tested, and will
provide an unprecedented overview of the majority of nuclease-mediated events per microinjection regime
tested, including allele frequency, i.e., HDR vs. NHEJ vs. wild type. The approach is not impacted by
downstream variables, including attrition to live born, or complicated by individual animals' mosaicism. This will
reduce costs per microinjection regime tested to <10% vs. that for live-born or fetus-derived data. Powerfully,
the approach allows comparative evaluation of 10-15 microinjection regimes simultaneously, a feat nearly
impossible when developing zygotes to fetuses or term, due to low survival, high costs, and time constraints.
We will: (Aim 1) Establish and validate, using preimplantation mouse embryos, a HTS-based analytical
strategy that precisely determines the targeted allele frequency and accuracy of targeted nuclease-mediated
DNA repair events, including HDR, for three selected genomic regions; (Aim 2) Test the principal variables
involved in targeted nuclease-mediated HDR, with a focus on DNA format, using the platform developed in Aim
1. For both aims and all three targeted regions, the optimal microinjection regime will be validated by repeating
the regime and bringing zygotes to term, followed by determination of germline transmission frequency. This
overall approach will provide a deeper understanding of the control of HDR vs. NHEJ in mouse zygotes,
allowing extensive, controlled manipulation of the mouse genome and potentially that of other species, leading
to a greater understanding of human disease via efficient manipulation of the mammalian genome.