Biology subjects

Yeung, A. T.

Publications and source records attributed to Yeung, A. T..

5 recordsLinked to original sources

bioRxiv2025

Harnessing anti-CRISPR to suppress, subtract, and segregate Cas9 activity for precision CRISPR in Drosophila

Tissue-specific CRISPR (ts-CRISPR) is a powerful approach for studying cell and developmental biology by restricting mutagenesis to specific tissues. However, the precision of this approach is often compromised by non-specific, "leaky" Cas9 activity that confounds phenotypic analysis and destabilizes Cas9/gRNA stocks. To address these limitations in Drosophila, we developed a toolkit based on the Anti-CRISPR (Acr) protein AcrIIA4. We first identified AcrIIA4 as a potent in vivo Cas9 inhibitor with high stability and established the temporal requirements for its function. Based on these findings, we generated three classes of Acr tools. First, a collection of AcrIIA4-bearing balancers robustly suppresses Cas9 and enables the stable maintenance of complex Cas9/gRNA stocks. Importantly, maternal deposition of AcrIIA4 from these balancers provides a means of temporal control, delaying Cas9 activity until metamorphosis. Second, tissue-specific AcrIIA4 transgenes refine leaky Cas9 drivers in a "tissue-subtraction" strategy. Finally, germline-specific and soma-specific Acr tools efficiently segregate Cas9 activity, solving bidirectional leakiness between these two compartments. This comprehensive AcrIIA4 toolkit provides new levels of precision, versatility, and temporal control for Drosophila CRISPR applications.

genetics↗

bioRxiv2025

Tuning Mitotic Recombination with Patterned DNA Nicks for Precision Mosaic Analysis

CRISPR/Cas9-based mosaic analysis is a powerful tool for in vivo genetics but is limited by cytotoxicity and mutagenesis associated with DNA double-strand breaks (DSBs). Here, we establish Cas9-derived nickases as safer and more reliable alternatives for inducing mitotic recombination in Drosophila. We demonstrate that single-strand nicks are sufficient to generate mosaic clones and systematically dissect the parameters governing this process. We find that clone frequency can be controlled by the gRNA nicking pattern, with two distant nicks on the same DNA strand synergistically enhancing recombination by over nine-fold compared to a single nick. Based on these findings, we propose a mechanistic model for nick-induced crossover and provide a versatile toolkit for generating tissue-specific nickases. This work establishes nickase-based MAGIC as a superior method for high-fidelity clonal analysis, enabling more precise investigation of gene function in development and disease. SIGNIFICANCE STATEMENTThe CRISPR/Cas9-based mosaic technique, MAGIC, is a versatile tool for in vivo biological investigations. However, its reliance on DNA double-strand breaks (DSBs) can cause significant, unintended cell damage. Here we establish that Cas9-derived nickases, which create gentler single-strand nicks, are a superior alternative. We show that nickases safely induce genetic mosaics in Drosophila by avoiding this cellular toxicity. By systematically dissecting the process, we discovered principles of gRNA design that allow clone frequencies to be tuned for different experimental needs. This work provides a new mechanistic model for nick-induced genetic exchange, a high-fidelity "nickase-MAGIC" method, and a versatile toolkit for precision clonal analysis.

developmental biology↗

bioRxiv2025

A genome-wide MAGIC kit for recombinase-independent mosaic analysis in Drosophila

Mosaic analysis has been instrumental in advancing developmental and cell biology. Most current mosaic techniques rely on exogenous site-specific recombination sequences that need to be introduced into the genome, limiting their application. Mosaic analysis by gRNA-induced crossing-over (MAGIC) was recently developed in Drosophila to eliminate this requirement by inducing somatic recombination through CRISPR/Cas9-generated DNA double-strand breaks. However, MAGIC has not been widely adopted because gRNA-markers, a required component for this technique, are not yet available for most chromosomes. Here, we present a complete, genome-wide gRNA-marker kit that incorporates optimized designs for enhanced clone induction and more effective clone labeling in both positive MAGIC (pMAGIC) and negative MAGIC (nMAGIC). With this kit, we demonstrate clonal analysis in a broad range of Drosophila tissues, including cell types that have been difficult to analyze using recombinase-based systems. Notably, MAGIC enables clonal analysis of pericentromeric genes and deficiency chromosomes and in interspecific hybrid animals, opening new avenues for gene function study, rapid gene discovery, and understanding cellular basis of speciation. This MAGIC kit complements existing systems and makes mosaic analysis accessible to address a wider range of biological questions. IMPACT STATEMENTA comprehensive toolkit enables genome-wide, recombinase-independent mosaic analysis in Drosophila, permitting clonal analysis of pericentromeric genes, deficiency chromosomes, and interspecific hybrids previously inaccessible to standard methods.

genetics↗

bioRxiv2024

Phagocytosis-driven neurodegeneration through opposing roles of an ABC transporter in neurons and phagocytes

Lipid homeostasis is critical to the survival of neurons. Lipid transporters from the ATP-binding cassette A (ABCA) subfamily are important regulators of lipid trafficking and are associated with multiple neurodegenerative diseases. How ABCA transporters regulate specific aspects of lipid homeostasis to impact neurodegeneration is an outstanding question. Here we report that the Drosophila ABCA protein Engulfment ABC Transporter in the ovary (Eato) contributes to phagocytosis-dependent neurodegeneration by playing two opposing roles in neurons and nearby phagocytes: In neurons, Eato prevents dendrites and axons from being attacked and engulfed by neighboring phagocytes; in phagocytes, however, Eato enhances the ability of these cells to detect neurons as engulfment targets. Thus, Eato deficiency in neurons alone results in severe phagocytosis-dependent dendrite and axon degeneration, whereas removing Eato from both neurons and phagocytes completely rescues the neurite degeneration. Surprisingly, Eato exerts its functions in both neurons and phagocytes by suppressing the effects of the eat-me signal phosphatidylserine (PS) exposed on the cell surface. Interestingly, multiple human and C. elegans ABCA homologs can compensate for the loss of Eato in phagocytes but not in neurons, suggesting both conserved and cell type-specific activities of these ABCA proteins. These results reveal how ABCA proteins participate in neurodegeneration by regulating PS homeostasis and imply possible mechanisms of neuron-phagocyte interactions in neurodegenerative diseases.

neuroscience↗

bioRxiv2022

A toolkit for converting Gal4 into LexA and Flippase transgenes in Drosophila

Drosophila has been a powerful model system for biological studies due to the wide range of genetic tools established for it. Among these tools, Gal4 is the most abundant, offering unparalleled tissue- and developmental stage-specificity for gene manipulation. In comparison, other genetic reagents are far fewer in choices. Here we present a genetic toolkit for converting Gal4 strains into LexA and Flippase transgenes through simple genetic crosses and fluorescence screening. We demonstrate the proof-of-principle by converting ten Gal4 lines that exhibit diverse tissue specificities and examined the activity patterns of the converted LexA and Flippase lines. Gal4-to-LexA and Flp conversion is fast and convenient and should greatly expand the choices of LexA and Flp for binary expression and FRT-based mosaic analysis, respectively, in Drosophila.

genetics↗