CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function

Mounting evidence implicates TDP-43 dysfunction and the accumulation of pathological cryptic exons across multiple neurodegenerative diseases, underscoring the need for accessible tools to detect and quantify TDP-43 loss-of-function (LOF). These tools are crucial for assessing potential disease contributors and exploring therapeutic candidates in TDP-43 proteinopathies. Here, we develop a sensitive and accurate real-time sensor for TDP-43 LOF: the CUTS (CFTR UNC13A TDP-43 Loss-of-Function) system. This system combines UG-rich sequences and previously reported cryptic exons regulated by TDP-43 with a reporter, enabling the tracking of TDP-43 LOF through live microscopy and RNA/protein-based assays. We show that CUTS effectively detects TDP-43 loss of function arising from mislocalization, impaired RNA binding, and pathological aggregation. Our results show the sensitivity and accuracy of the CUTS system in detecting and quantifying TDP-43 LOF, opening avenues to explore unknown TDP-43 interactions that regulate its function. In addition, by replacing the fluorescent tag in the CUTS system with the coding sequence for TDP-43, we show significant recovery of its function under TDP-43 LOF conditions, highlighting the potential utility of CUTS for self-regulating gene therapy applications. In summary, CUTS represents a platform for evaluating TDP-43 LOF in real-time and gene-replacement therapies in neurodegenerative diseases associated with TDP-43 dysfunction. HighlightsO_LICUTS is a cryptic exon RNA biosensor enabling real-time detection of TDP-43 loss of splicing function. C_LIO_LICUTS exhibits a linear relationship with a reduction in TDP-43 protein. C_LIO_LICUTS can deliver an autoregulated gene payload in response to TDP-43 loss-of-function. C_LIO_LITDP-43 homotypic phase transitions and cell stress induce loss of splicing function detected via CUTS. C_LI