Presentation description

CRISPR-Cas9 is a powerful genome-editing tool, yet its application in whole-organism genetic screens is limited by time and throughput. Our lab has developed Multiplexed Intermixed CRISPR Droplet (MIC-Drop), a new technology that combines CRISPR-Cas9 with microfluidics and gene-specific DNA barcoding to systemically mutagenize hundreds-thousands of genes in zebrafish (Danio rerio). We utilize zebrafish within our study because of their advantages as a model organism: genetic tractability, high fecundity, and amenability to high-throughput screening. MIC-Drop allows the evaluation of morphological changes and identification of the causal gene via DNA barcode sequencing; within our current study, we plan to expand on this platform to develop MIC-Drop-metabolomics. By replacing the DNA barcode with an isotope-labeled, small-molecule barcode, MIC-Drop screens can be integrated with liquid chromatography-mass spectrometry-based metabolomics readout, yielding detailed metabolome data for each mutagenized embryo and revealing the associated gene in the same analysis. To test this project's feasibility, we conducted preliminary experiments to answer whether metabolites can be detected from single zebrafish embryos, and how long the injected metabolite barcode persists in vivo. We found that major metabolites such as amino acids were indeed detectable from individual embryos and confirmed barcode (alanine-D4) stability up to at least 2 days-post-fertilization. With this, we developed a proof-of-concept mini-screen targeting four genes associated with inborn errors of metabolism (dlat, dbt, pah, and ctns). Within the mini-screen, five CRISPR target sequences were selected for each gene using the CHOPCHOP software. DNA template generation, guide RNA synthesis, and barcoded droplet production were successfully completed for dlat. Our future plans include continued droplet generation, microinjection, metabolomic analysis, and expansion toward larger gene sets. In summary, MIC-Drop-metabolomics is emerging as a transformative approach with promising implications for metabolic studies.