Identifying the genetic causes of stillbirth using C. elegans as an in vivo platform

About This Grant

Project Summary: Stillbirth is defined as fetal death in utero past 20 weeks of gestation1. While it accounts for 60% of perinatal deaths, it remains unexplained in 25-60% of cases1. Although advances in genetic testing have revealed a genetic cause for approximately 20% of cases, there remains a large swath of unexplained stillbirths, especially for phenotypically complex missense mutations in the form of single nucleotide variants (SNVs)2. New in silico analyses and fetal sequencing technologies have enabled better capture of these SNVs as well as prediction of deleteriousness using constraints metrics3. However, these tools all rely on predictive modelling, leaving a wide gap in our understanding of the in vivo function of these variants, and thereby our ability to use them as diagnostics in prenatal testing. Thus, there exists a critical need for a high throughput in vivo platform capable of discriminating between deleterious and benign SNVs originating from fetal exome data. The nematode Caenorhabditis elegans serves as an ideal chassis for this platform because of its scalability, genetic tractability, and most relevant, its 83% proteome orthology4. I intend to employ this model to develop a high throughput platform that can test SNVs from fetal exome data by leveraging multiplexed CRISPR/Cas technology. My preliminary evidence demonstrates that a pipeline for taking human genes and SNVs to test them in the worm is feasible and scalable. The overarching goal of this proposal is to use multiple cutting-edge parallelized CRISPR methods to generate worm humanization platforms capable of assessing SNVs involved in fetal demise through three aims. The first aim will be to produce a humanized worm collection for the top 100 most well conserved FD genes using extant multiplexed CRISPR insertion in a safe harbour landing site. The second will be to adapt pre-existing parallelized CRISPR methods to generate customized landing sites for all fetal demise genes that will then go on to serve as the background strain to test every allele of that gene. The third aim will leverage an error-prone DNA polymerase-nCas9 construct for precision mutagenesis across fetal demise genes enabling me to evaluate fetal demise alleles at scale. My central hypothesis is that C. elegans can be used as a scalable model to determine the function of human SNVs involved in fetal demise. Completion of these studies will yield a unique resource capable of modelling all fetal demise genes and their SNVs, which will eventually allow for better prenatal diagnostics. In tandem, this proposal will also serve to jumpstart my aspirations towards a career as an interdisciplinary scientist through intensive training in C. elegans genetics, functional genomics, informatics, genetic engineering, and high throughput approaches coupled with a star-studded, multi-institutional guidance and mentorship plan.