Crispr interference to identify HSA21 genes reducing neurogenesis - down syndrome (DS) is caused by complete or partial trisomy of human chromosome 21 (HSA21)

The University of Texas Southwestern Medical Center received a $451,000 Project Grant award from the National Institutes of Health (NIH) Office of the Director under the Trans-NIH Research Support program (CFDA 93.310), effective July 1, 2025 through June 30, 2027. This R21 exploratory grant funds research to develop CRISPR interference (CRISPRi) technology tools and high-throughput screening methodologies for identifying genes on human chromosome 21 (HSA21) that contribute to Down syndrome pathologies. The research leverages induced pluripotent stem cells (iPSCs) derived from individuals with Down syndrome to conduct systematic gene repression studies, with the objective of identifying molecular targets for potential therapeutic intervention.

The project deliverables include development and integration of a CRISPR-based gene-silencing system into Down syndrome iPSCs, design of optimized guide RNAs for graded gene repression, and establishment of screening platforms that can detect and quantify key cellular phenotypes associated with Down syndrome at the single-cell level. Specifically, the research targets two distinguishing cellular characteristics: elevated mitochondrial membrane potential in pluripotent Down syndrome cells and reduced capacity for neuroectodermal lineage commitment, the latter being directly relevant to intellectual disability. By establishing these molecular screening tools, the award aims to remove a significant roadblock in identifying genes responsible for Down syndrome's complex phenotype and facilitate development of targeted therapeutic approaches to improve outcomes for affected individuals.

IT comprises a complex phenotype of over 80 features including metabolic dysregulation and a marked reduction in both neurogenesis and brain size. The specific molecular mechanisms by which trisomy of HSA21 gives rise to DS pathologies remain unknown. Identifying the dominant genes or molecular pathways involved would open the possibility for developing targeted therapies to improve the lives of affected individuals. Here, we propose combining crispr interference (crispri) technology with induced pluripotent stem cells (ipscs) derived from individuals with DS (3S-ipscs) to develop a selectable screen for HSA21 genes that contribute to DS pathologies. The complexity of the DS phenotype has been a roadblock to definitive identification of genes that play prominent roles in the syndrome. Our preliminary data show evidence of two important, fundamental differences between DS cells and isogenic, euploid controls that can be detected and quantified at the single-cell level, thereby opening the door widely for the development of high through-put screens for gene identification. One of these key differences, elevated mitochondrial membrane potential (∆ΨM), is evident in 3S-ipscs in the pluripotent state; the other, reduced ability to commitment to a neuroectodermal (NE) lineage, is directly relevant to what is arguably the most prominent feature of DS, that of intellectual disability. The goal of this R21 proposal is to build the tools necessary to carry out crispri-based, high throughput screens to identify genes on HSA21 that contribute to DS pathologies at the earliest stages of development. Aim 1 will use talen-nuclease directed editing to integrate a transgene expressing DCAS9-krab into a safe-harbor locus in 3S-ipscs. Aim 2 will focus on designing guide rnas that achieve graded repression to more closely model a reduction in gene dosage from the trisomic three copies to the normal two copies. Successful completion of our proposed studies will provide proof-of-concept for our approach to functional gene identification in DS and lay the foundation for expanding to comprehensive and combinatorial screens of the full coding and non-coding content of HSA21. IT will also make a powerful and flexible tool available to the DS research community due to the totipotency of ipscs.