Biology subjects

Bracha, S.

Publications and source records attributed to Bracha, S..

3 recordsLinked to original sources

bioRxiv2026

Library transgenesis in zebrafish through delayed site-specific mosaic integration for in vivo pooled screening of transgenes

Functional screening through systematic deletion, editing or addition of libraries of genes is a powerful approach for discovering gene functions and developing improved molecular tools. However, due to the need for high throughput, such campaigns are typically conducted in vitro, leading to many discoveries, especially tools and therapeutics, which fail to translate in vivo. Tissue context, cellular physiology, and systemic regulation shape both tool performance and gene function in ways that simplified culture systems cannot predict. Pooled in vivo screening methods have the potential to enable screening within living animals while preserving the physiological context, but current approaches using viral vectors face three critical limitations: multi-transgene insertions per cell confound genotype-phenotype association, viral packaging constrains transgene size, and cell-type tropism restricts and biases targeting. Here, we introduce a zebrafish library transgenesis method that overcomes these limitations through delayed site-specific mosaic integration. We exploit a temporal delay between library microinjection with PhiC31 mRNA, and library integration, to allows the library to spread episomally throughout the developing embryo before integration begins. This produces mosaic animals where each cell independently integrates one randomly-selected library member, enforced by a single genomic AttP landing site. We demonstrate delivery of multi-kilobase transgenes with high library coverage of 1,378-1,989 unique integrants per animal, and single-transgene-per-cell in [~]99% of brain cells. This method provides a platform for direct in vivo screening of large transgene libraries with single-transgene precision, with potential applications in both biological discovery and tool development.

bioengineering↗

bioRxiv2025

Screening channelrhodopsins using robotic intracellularelectrophysiology and single cell sequencing

BackgroundOur ability to engineer opsins is limited by an incomplete understanding of how sequence variations influence function. The vastness of opsin sequence space makes systematic exploration difficult. New methodIn recognition of the need for datasets linking opsin genetic sequence to function, we pursued a novel method for screening channel-rhodopsins to obtain these datasets. In this method, we integrate advances in robotic intracellular electrophysiology (Patch) to measure optogenetic properties (Excite), harvest individual cells of interest (Pick) and subsequently sequence them (Sequence), thus tying sequence to function. ResultsWe used this method to sequence more than 50 cells with associated functional characterization. We further demonstrate the utility of this method with experiments on heterogeneous populations of known opsins and single point mutations of a known opsin. Of these point mutations, we found C160W ablates ChrimsonRs response to light. Conclusion and comparison to existing methodsCompared to traditional manual patch clamp screening, which is labor-intensive and low-throughput, this approach enables more efficient, standardized, and scalable characterization of large opsin libraries. This method can enable opsin engineering with large datasets to increase our understanding of opsin sequence-function relationships.

neuroscience↗

bioRxiv2018

Engineering Brain Parasites for Intracellular Delivery of Therapeutic Proteins

Protein therapy has the potential to alleviate many neurological diseases; however, delivery mechanisms for the central nervous system (CNS) are limited, and intracellular delivery poses additional hurdles. To address these challenges, we harnessed the protist parasite Toxoplasma gondii, which can migrate into the CNS and secrete proteins into cells. Using a fusion protein approach, we engineered T. gondii to secrete therapeutic proteins for human neurological disorders. We tested two secretion systems, generated fusion proteins that localized to T. gondiis secretory organelles and assessed their intracellular targeting in various mammalian cells including neurons. We show that T. gondii expressing GRA16 fused to the Rett syndrome protein MeCP2 deliver a fusion protein that mimics the endogenous MeCP2, binding heterochromatic DNA in neurons. This demonstrates the potential of T. gondii as a therapeutic protein vector, which could provide either transient or chronic, in situ synthesis and delivery of intracellular proteins to the CNS.

synthetic biology↗