Three synthetic gRNAs per gene achieve over 90% biallelic knockouts in F0. (A) Schematic of the F0 knockout strategy. Introduction of indels at multiple loci within the target gene leads to frameshift and premature stop codons and/or mutation of essential residues. (B) Simplified theoretical model of biallelic knockout probability as a function of number of targeted loci, assuming frameshift is the sole knockout mechanism. PKO, probability of biallelic knockout; Pmutation, mutation probability (here, 1.00 or 0.80); Pframeshift, probability of frameshift after mutation (0.66); nloci, number of targeted loci. (C–D) (top) Phenotypic penetrance as additional loci in the same gene are targeted. Pictures of the eye at 2 dpf are examples of the scoring method. (bottom) Unviability as percentage of 1-dpf embryos. (E–F) Proportion of alleles harbouring a frameshift mutation if 1, 2, 3, or 4 loci in the same gene were targeted, based on deep sequencing of each targeted locus. Each line corresponds to an individual animal. (G) 2.5-month wild-type and slc24a5 F0 knockout adult fish (n = 41). (H) 2-dpf progeny from slc24a5 F0 adults outcrossed to wild types (n = 283) or incrossed (n = 313). (I) Example of 3-dpf wild type, mab21l2u517 mutant, and mab21l2 F0 embryos (n = 96/100 injected). See also Figure 1—figure supplement 1.
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