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Mic drop sentence5/29/2023 ![]() ![]() Embryos with gross morphological defects at 3 dpf (~15%) were removed and the barcodes of those with cardiac defects were sequenced. ( D) MIC-Drop screen of the 188 candidate genes and subsequent phenotyping shows no significant differences in viability between uninjected and droplet-injected embryos by 3 dpf N = (Left panel: Uninjected-1801, Injected-2502 Right panel: Uninjected-1571, Injected-2013). ( C) gRNA sequences with fewer off-targets were prioritized. ( B) Filtering to remove genes with known mutant phenotypes yields 188 poorly-characterized genes potentially important for cardiovascular development in zebrafish. ~14% of the genes (orange dots) have reported cardiac phenotypes in ZFIN suggesting enrichment of genes important in heart development. (A) A publicly available dataset was used to populate a list of candidate genes enriched in the embryonic zebrafish heart. Inset: Hundreds of pseudo-colored droplets (used as proxies for droplets targeting different genes) do not fuse when transferred to an injection needle. Total N sequenced = 230 from 3 separate injections. ( E) Single-needle injection of intermixed droplets targeting three different genes ( tyr, tnnt2a, chrd) and subsequent phenotyping shows even representation of each droplet with all of the embryos exhibiting only one of the three expected phenotypes, and none (0%) were wildtype. a: Uninjected b: Traditional RNP injection c: MIC-Drop injection. ( D) RNP-containing droplets are non-toxic and stable for prolonged storage – retaining activity at least 28 days of storage at 4☌. ( C) Injection of droplets containing RNPs targeting tyr, rx3, tbx5a, and chrd genes recapitulates known mutant phenotypes in F0, highlighted by red boxes. Embryos showing phenotypes-of-interest are isolated and the causative genotype is identified by retrieving and sequencing the barcode. ![]() Droplets targeting multiple genes are intermixed, loaded into a single injection needle and injected serially into one-cell zebrafish embryos. A microfluidics device generates nanoliter-sized droplets, each containing ribonucleoproteins (RNP) targeting a gene-of-interest and a unique DNA barcode associated with the gene.
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