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Acute multi-sgRNA CRISPR/Cas9 knockout reproduces the microcephaly phenotype of stable osgep and tprkb KO lines. Larvae were imaged from a dorsal view. Head diameter to total body length was calculated. This ratio was normalized by the mean of the uninjected/wildtype control and defined as “microcephaly index” (MI). MI was determined at 6 dpf except for the stable KO line osgep c.102_105del at 4 dpf due to early lethality (see Fig 4a). (a) Representative phenotypes of a larva that was injected with the osgep targeting multi-sgRNA pool compared to a scrambled control larva. The red lines display the typical axes used for the measurements. (b) For osgep, the acute multi-sgRNA KO showed a significant microcephaly compared to uninjected and scrambled control. (c) The microcephaly phenotype is recapitulated in the homozygous larvae of the stable KO line osgep c.102_105del compared to wildtype and heterozygous clutch mates. (d) For osgep, MI is significantly higher in acute KO larvae compared to homozygous stable KO larvae. (e) For tprkb, the acute multi-sgRNA KO showed a significant microcephaly compared to uninjected and scrambled control. (f) The microcephaly is recapitulated in the homozygous larvae of the stable KO line tprkb c.370_376delinsAA compared to wildtype and heterozygous clutch mates. (g) For tprkb, no significant difference in MI was found for acute KO larvae compared to homozygous stable KO larvae