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bioRxiv · 10.1101/2025.05.27.656468

MIC-Drop-seq: Scalable single-cell phenotyping of mutant vertebrate embryos

Carey, C. M.·Parvez, S.·Brandt, Z. J.·Bisgrove, B. W.·Yates, C. J.·Peterson, R. T.·Gagnon, J. A.
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Abstract

Advances in genome engineering and single-cell RNA sequencing (scRNAseq) have revolutionized the ability to precisely map gene functions, yet scaling these techniques for large-scale genetic screens in animals remains challenging. We combined high-throughput gene disruption in zebrafish embryos via Multiplexed Intermixed CRISPR Droplets with phenotyping by multiplexed scRNAseq (MIC-Drop-seq). In one MIC-Drop-seq experiment, we intermixed and injected droplets targeting 50 transcriptional regulators into 1,000 zebrafish embryos, followed by pooled scRNAseq. Tissue-specific gene expression and cell abundance analysis of demultiplexed mutant cells recapitulated many known phenotypes, while also uncovering novel functions in brain and mesoderm development. We observed pervasive cell-extrinsic effects among these phenotypes, highlighting how whole-embryo sequencing captures complex developmental interactions. Thus, MIC-Drop-seq provides a powerful and scalable platform for mapping gene functions in vertebrate development with cellular resolution.

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Carey, C. M., Parvez, S., Brandt, Z. J., Bisgrove, B. W., Yates, C. J., Peterson, R. T., Gagnon, J. A.. 2025-05-29. MIC-Drop-seq: Scalable single-cell phenotyping of mutant vertebrate embryos. https://doi.org/10.1101/2025.05.27.656468

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