Advances in high-throughput sequencing (HTS) have accelerated the discovery of the genetic basis of
numerous human diseases and conditions, including congenital disorders. These conditions affect a wide
range of physiological systems, including cardiovascular, craniofacial, nervous, genital/urinary, in addition to
general defects growth and dysmorphology. However, the process of ascribing causality to a given variant can
be challenging and remains a major bottleneck in our understanding of human disease. Animal model
validation is a powerful tool to evaluate the causality of a given variant, and the mouse provides the ideal
mammalian system to model developmental disorders. CRISPR/Cas9 technology has simplified and reduced
the timelines for mouse model creation, but breeding time will still typically take up to a year or more before
phenotypes can be examined. Thus, a validation platform that directly examines this founder population (F0
generation) promises to massively reduce the time and expense required for validation of new human disease
mutations in the mouse. Our preliminary work provides proof-of-principle evidence that this approach is
feasible, however challenges remain before such an approach can be applied to a wide range of human
developmental conditions. Direct screening of F0 embryos provides a means to examine more difficult
engineering and breeding challenges, include dominant mutations that are predicted to be lethal in the mouse,
and modeling multigenic causes of disease. Moreover, a F0 platform can provide a tool for rapid, direct genetic
interrogation of pathways through multiplex mutagenesis. Challenges remain, however, before this approach
can be effectively implemented as a robust platform. For example, while generation indels and deletions
through error-prone non-homologous end joining (NHEJ) repair is highly efficient, editing of specific mutations
through homology directed repair (HDR) occurs at a much lower rate. Additionally, founders and F0 embryos
are typically mosaic and the tools to quantitatively assess both the spectrum of alleles and the mutagenesis
rate for each require further development. Therefore, the overarching goal of this proposal is to build upon our
proof-of-principle studies to optimize our F0 screening platform and implement it to model structural birth
defects. We will test our hypotheses that 1) modification of parameters of the CRISPR mutagenesis protocol
can improve the efficiency and reduce the mosaicism of HDR; and 2) that we can apply the platform to specific
unique congenital disease challenges not easily modeled with standard mouse genetics approaches. This will
take advantage of several key resources, including a high-throughput embryonic phenotyping pipeline
developed for the Knockout Mouse Phenotyping Program (KOMP2) and the large-scale mouse production
capabilities of The Jackson Laboratory. The long-term goal is to integrate our screening platform with large-
scale precision modeling initiatives, providing a rapid and robust means to evaluate new genetic variants in a
mammalian model system.