Biology subjects

KC, R.

Publications and source records attributed to KC, R..

6 recordsLinked to original sources

bioRxiv2025

Development of a point-of-care field diagnostic test for DFT1 and DFT2

The Tasmanian devil (Sarcophilus harrisii) population has undergone a major decline in the wild due to the epidemics of two transmissible cancers known as devil facial tumours 1 (DFT1) and devil facial tumour 2 (DFT2). Understanding the distribution and prevalence of DFT1 and DFT2 is challenging as they can only be detected in field conditions when tumours are large enough to be visually observed. This impedes our ability to understand how the devil population is evolving in response to the tumours. PCR-based diagnostics are available, but the technique is not field-applicable. To overcome these hurdles, we developed an ultrasensitive and field-deployable rapid diagnostic tool. We used a technique called Specific High-Sensitivity Enzymatic Reporter Unlocking (SHERLOCK), which combines an isothermal nucleic acid amplification step with CRISPR-based recognition and activation of reporter probes to indicate target sequence detection. This system requires only portable laboratory equipment, making it more amenable to field conditions. The results showed that our test can detect DFT1 and DFT2 DNA within the attomolar range. We then confirmed that the test can detect DFT1 in the field using non-invasive swab samples. The SHERLOCK test can be used for more active monitoring and management approaches that are not currently possible.

molecular biology↗

bioRxiv2025

Exploring Prime Editing strategies for genetic correction of a Ceroid Neuronal Lipofuscinosis type 2 disease causing variant

Neuronal ceroid lipofuscinosis type 2 (CLN2) is a rare, fatal paediatric neurodegenerative genetic disease that causes progressive psychomotor decline, epilepsy, speech impairment, vision loss, and premature death by late childhood or early adolescence. It is caused by mutations in the TPP1 gene, which encodes the tripeptidyl peptidase 1 enzyme. To date, enzyme replacement therapy (ERT) with recombinant human TPP1 is the only approved treatment available and it does not appear to have a curative effect on disease progression or rescuing complex motor functions. This underscores the importance of developing more effective treatment strategies for CLN2 disease. In the quest for an alternative therapy, we here seek to utilise CRISPR based prime editing (PE) to correct one of the most frequent TPP1 mutations of c.622 C>T (p. Arg208Ter). In cultured mammalian HEK293a cells we screened 16 different PE and pegRNA combinations, achieving on-target editing efficiencies up to 27.36 %. To enable efficient in vivo gene editing, we packaged the most promising PE and pegRNA combination using engineered virus-like particles (eVLPs) which resulted in 12.6 % on-target editing. Given the robust on-target editing and reduced risk of bystander editing, PE is deemed suitable for in vivo testing in the pre-clinical models for CLN2 disease. Overall, our findings establish the proof of concept for CRISPR-based prime editing to correct pathogenic human TPP1 nonsense mutation of c.622 C>T (p. R208X) and provide a basis for further investigations of PE as a genetic treatment for CLN2 disease.

molecular biology↗

bioRxiv2025

Systematic comparison of Generative AI-Protein Models reveals fundamental differences between structural and sequence-based approaches.

Recent advances in artificial intelligence have led to the development of generative models for de novo protein design. We compared 13 state-of-the-art generative protein models, assessing their ability to produce feasible, diverse, and novel protein monomers. Structural diffusion models generally create designs with higher confidence in predicted structures and more biologically plausible energy distributions, but exhibit limited diversity and strong sequence biases. Conversely, protein language models generate more diverse and novel designs but with lower structural confidence. We also evaluated these models ability to generate unique proteins, conditionally based on the Tobacco Etch Virus (TEV) protease. Generative models were successful in producing functional enzymes, albeit with diminished activity compared to the wildtype TEV. Our systematic benchmarking provides a foundation for evaluating and selecting generative protein models, while highlighting the complementary strengths of different generative paradigms. This framework will facilitate an informed application of these tools for bio-medical engineering and design.

synthetic biology↗

bioRxiv2024

Multimodal Hox5 activity generates motor neuron diversity

Motor neurons (MNs) are the final output of circuits driving fundamental behaviors, such as respiration and locomotion. Hox proteins are essential in generating the MN diversity required for accomplishing these functions, but the transcriptional mechanisms that enable Hox paralogs to assign distinct MN subtype identities despite their promiscuous DNA binding motif are not well understood. Here we show that Hoxa5 controls chromatin accessibility in all mouse spinal cervical MN subtypes and engages TALE co-factors to directly bind and regulate subtype-specific genes. We identify a paralog-specific interaction of Hoxa5 with the phrenic MN-specific transcription factor Scip and show that heterologous expression of Hoxa5 and Scip is sufficient to suppress limb-innervating MN identity. We also demonstrate that phrenic MN identity is stable after Hoxa5 downregulation and identify Klf proteins as potential regulators of phrenic MN maintenance. Our data identify multiple modes of Hoxa5 action that converge to induce and maintain MN identity.

developmental biology↗

bioRxiv2022

Coordinated cadherin functions sculpt respiratory motor circuit connectivity

Breathing, and the motor circuits that control it, are essential for life. At the core of respiratory circuits are Dbx1-derived interneurons, which generate the rhythm and pattern of breathing, and phrenic motor neurons (MNs), which provide the final motor output that drives diaphragm muscle contractions during inspiration. Despite their critical function, the principles that dictate how respiratory circuits assemble are unknown. Here we show that coordinated activity of a type I cadherin (N-cadherin) and type II cadherins (Cadherin-6, -9, and -10) is required in both MNs and Dbx1-derived neurons to generate robust respiratory motor output. Both MN- and Dbx1-specific cadherin inactivation during a critical developmental window results in perinatal lethality due to respiratory failure and a striking reduction in phrenic MN bursting activity. This combinatorial cadherin code is required to establish phrenic MN cell body and dendritic topography; surprisingly, however, cell body position appears to be dispensable for the targeting of phrenic MNs by descending respiratory inputs. Our findings demonstrate that type I and type II cadherins function cooperatively throughout the respiratory circuit to generate a robust breathing output and reveal novel strategies that drive the assembly of motor circuits.

neuroscience↗

bioRxiv2021

ASXL3 controls cortical neuron fate specification through extrinsic self-renewal pathways

During corticogenesis, transcription plasticity is fundamental to the restriction of neural progenitor cell (NPC) multipotency and production of cortical neuron heterogeneity. Human and mouse genetic studies have highlighted the role of Polycomb transcriptional regulation in this process. ASXL3, which encodes a component of the Polycomb repressive deubiquitination (PR-DUB) complex, has been identified as a high confidence autism spectrum disorder (ASD) risk gene. Genetic inactivation of Asxl3, in a mouse model that carries a clinically relevant ASXL3 frameshift (Asxl3fs) variant, disrupts lateral expansion of NPCs and delays cortical neuron differentiation. Single-cell RNA sequencing analysis implicates Notch signaling, which alters the composition of excitatory neurons and fidelity of cortical layer deposition. Our data provides a new link between extrinsic signaling cues and intrinsic epigenetic regulation that together control the timing of cell fate programs. Furthermore, transcriptomic analysis revealed dysregulation of other known ASD risk genes indicating that a convergent developmental pathway is affected. Collectively our work provides important insights about developmental mechanisms that contribute to ASD neuropathology.

neuroscience↗