Embryos were injected with 300 pg of Cas9 mRNA and 50 pg of each sgRNA at one-cell stage using a PicoPump (World Precision Instruments) and standard microinjection protocol (29 ). The amount of sgRNA and Cas9 mRNA for each injection was calculated by CRISPR-CALC (Supplementary File S1). For majority of the sgRNAs, we mixed sgRNAs to two genes at 50 pg each to reduce the number of injections. To test for multiplexing, we mixed eight sgRNAs at 25 pg each with 300 pg of Cas9 mRNA (Supplementary File S1). Injected embryos were incubated at 28.5°C and euthanized at 48 hpf for DNA extraction. DNA was extracted from eight uninjected and eight injected embryos for each sgRNA using Extract-N-Amp Tissue PCR Kit (Sigma) with one-fourth of the recommended volumes for each of the solutions. Extracted DNA was diluted at 1:10 ratio with ultra pure water and 1.5 μl was used as template for subsequent PCR reactions.