Biology subjects

Leem, E.

Publications and source records attributed to Leem, E..

3 recordsLinked to original sources

bioRxiv2026

CRISPR-Associated Transposases Enable Programmable DNA Integration in Plants

Programmable DNA integration is a major challenge in plant genome engineering. CRISPR-associated transposases (CAST) catalyze efficient RNA-guided DNA integration without double-strand breaks, yet their activity has not been established in plants. Here, we reconstituted and engineered a Type I-F CAST for programmable DNA integration in plant cells. We validated expression of the wild-type Pseudoalteromonas CAST (PseCAST) machinery in plants and established targeted episomal integration in Arabidopsis thaliana protoplasts and chromosomal integration at a transgenic locus in Nicotiana benthamiana. The evolved PseCAST system, evoCAST, showed chromosomal integration efficiencies of 2.7%, representing a 6-fold improvement over wild-type PseCAST. evoCAST also enabled the insertion of cis-regulatory elements into a synthetic landing pad with 8% efficiency. evoCAST was subsequently retargeted to six endogenous genomic loci, demonstrating programmable integration across diverse chromosomal contexts. Finally, a cofactor screen identified the chromatin-associated factor AtHMGB2 as an enhancer of evoCAST-mediated integration activity in plants. These results establish CAST as a functional platform for programmable DNA insertion in plants and provide a foundation for developing targeted genome-engineering technologies for crop biotechnology.

bioengineering↗

bioRxiv2024

Near infrared fluorescent nanosensors for high spatiotemporal oxytocin imaging

Oxytocin is a neuropeptide involved in regulating social and emotional behavior. Current techniques for oxytocin imaging are generally limited in spatial and temporal resolution, real-time imaging capacity, selectivity for oxytocin over vasopressin, and application in young and non-model organisms. To address these issues, we developed a method to evolve purely synthetic molecular recognition for oxytocin on the surface of near-infrared fluorescent single-walled carbon nanotubes (SWCNT) using single-stranded DNA (ssDNA). The best-performing nanosensor nIROT-SELEC reversibly undergoes up to a 172% fluorescence increase in response to oxytocin with micromolar dissociation, nanomolar limit of detection, and and high selectivity over oxytocin analogs, receptor agonists and antagonists, and co-released neurochemicals. We next demonstrated the versatility of nIROT-SELEC by performing live imaging of synaptic evoked oxytocin released in acute brain slices of mice and prairie voles. Our method for high throughput evolution of neuropeptide nanosensors holds promise to enable synaptic scale visualization of neuropeptide signaling in the brain cross different species and developmental stages, to advance the study of neurochemical signaling for its role in both health and disease.

neuroscience↗

bioRxiv2022

Near Infrared Nanosensors Enable Optical Imaging of Oxytocin with Selectivity over Vasopressin in Acute Mouse Brain Slices

Oxytocin plays a critical role in regulating social behaviors, yet our understanding of its role in both neurological health and disease remains incomplete. Real-time oxytocin imaging probes with the spatiotemporal resolution relevant to its endogenous signaling are required to fully elucidate oxytocin function in the brain. Herein we describe a near-infrared oxytocin nanosensor (nIROx), a synthetic probe capable of imaging oxytocin in the brain without interference from its structural analogue, vasopressin. nIROx leverages the inherent tissue-transparent fluorescence of single-walled carbon nanotubes (SWCNT) and the molecular recognition capacity of an oxytocin receptor peptide fragment (OXTp) to selectively and reversibly image oxytocin. We employ these nanosensors to monitor electrically stimulated oxytocin release in brain tissue, revealing oxytocin release sites with a median size of 3 m which putatively represents the spatial diffusion of oxytocin from its point of release. These data demonstrate that covalent SWCNT constructs such as nIROx are powerful optical tools that can be leveraged to measure neuropeptide release in brain tissue.

bioengineering↗