CRISPR KO screen to interrogate regulators of TDP-43 splicing

Summary: TDP-43 is a nucleic-acid binding protein that functions predominantly within the nucleus to promote proper RNA splicing and metabolism and is expressed across a variety of cell types. Disruption to TDP-43 splicing regulation and the associated loss-of-function (LOF) is observed in a variety of neurological disorders, including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD), and is often due to cytoplasmic mislocalization or nuclear condensation events. However, the exact mechanisms driving TDP-43 cytoplasmic mislocalization and compromised splicing regulation are not known. Identification of novel genetic interactors of TDP-43 and the biological pathways they are involved in which potentially influence TDP-43 splicing regulation can generate insight towards biological mechanisms to explore. Through investigating cellular dynamics implicated upstream of TDP-43 dysfunction, we can better understand early neurodegenerative disease mechanisms and thus brainstorm novel, viable therapeutic avenues in an attempt to mitigate disease pathogenesis in these disorders with TDP-43 "proteinopathies". To address this, we first developed a synthetic TDP-43 RNA splicing biosensor termed CUTS (CFTR-UNC13A TDP-43 loss-of-function sensor), which enables accurate detection of TDP-43 LOF using a GFP reporter construct based on previously characterized cryptic exon events that occur in contexts where TDP-43 splicing function has been disrupted. We then performed a genome-wide CRISPR knockout (KO) screen in HEK293 cells stably expressing the CUTS technology to investigate the genes and pathways implicated in TDP-43 LOF pathological cellular context. We hypothesize that unbiased genetic screening of factors that contribute to TDP-43 splicing functions or attempt to compensate for TDP-43 function in LOF contexts will uncover potential disease relevant genes and pathways. HEK293 cells with stable expression of the TDP-43 CUTS biosensor were transfected with the Brunello human genome sgRNA library at a low multiplicity of infection (MOI) so that a single sgRNA would be introduced per cell. Cells were FACS sorted into either GFP+ (TDP-43 LOF) or GFP- populations, ultimately yielding ~1% GFP+ cells. The resulting screening data were analyzed to determine positively and negatively selected genes within the GFP+ TDP-43 LOF cell population and resulting ranked gene hits were subsequently used to run pathway enrichment analyses. Within the TDP-43 LOF cell population, top enriched pathway hits from the negatively selected gene pool included processes implicated in neurodevelopment and chromatin organization, viral infection response signaling, and extracellular matrix organization. These results elucidate TDP-43 cellular interactors that may influence proper splicing regulation and inform targeted follow up investigations to explore novel therapeutic targets. Jessica Merjane, Longxin Xie, Cristian A. Bergmann, Jiazhen Xu, Bryan Hurtle, and Christopher J. Donnelly