The ability to inject all the intermixed droplets from a single needle circumvents the laborious and wasteful process of filling a separate needle for each gene targeted. Embryos are raised en masse, those exhibiting phenotype(s)-of-interest are isolated, and the identities of the perturbed genes are rapidly uncovered by retrieving and sequencing the barcodes.
Mic drop state movie#
Droplets targeting hundreds to thousands of different genes are intermixed together and injected into zebrafish embryos from a single needle ( Movie S1). The platform uses microfluidics to generate nanoliter-sized droplets, each droplet containing Cas9, multiplexed gRNAs targeting a gene-of-interest, and a unique barcode associated with each target gene. We have developed a platform, Multiplexed Intermixed CRISPR Droplets (MIC-Drop), for performing large-scale reverse-genetic screens in zebrafish ( Fig. Recent studies using multiplexed gRNAs to generate biallelic F0 mutants that successfully phenocopy germline mutant phenotypes are a welcome step but have not been scaled up for genome-wide CRISPR screens ( 12– 17). The largest such screen to date targeted 128 genes in zebrafish ( 10, 11). Targeting genes-of-interest is typically done one gene at a time-designing individual guide RNAs (gRNA), injecting Cas9-gRNA ribonucleoprotein (RNP) complexes, maintaining, propagating, and genotyping groups of fish-requiring extensive time, labor, and space. Reverse genetics approaches such as CRISPR have potential to circumvent some of the issues of forward genetics but are severely limited in throughput ( 8, 9). While impressive in scale, forward genetic techniques are time- and labor-intensive requiring years to link a desired phenotype with the genotype. These screens have proven invaluable in identifying key pathways regulating vertebrate development ( 3– 5) and behavior ( 6, 7). Historically, large scale genetic screens in zebrafish have employed forward genetic techniques such as chemical or insertional mutagenesis ( Fig.
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