Crispr/Cas9 Generation and T7 Endonuclease Assay

sgRNA for mavs was designed by using ChopChop (https://chopchop.cbu.uib.no/). sgRNA for slc45a2 (gene involved in pigmentation), sting and tnfa were previously designed and validated (8). Genotyping primers were designed by Primer3 (https://bioinfo.ut.ee/primer3-0.4.0/) and validated in USCS Genome Browser (https://genome.ucsc.edu/cgi-bin/hgPcr). sgRNAs were produced by sgRNA IVT kit (Takara Bio) by following the manufacturer's instructions and RNA was isolated by Trizol (Invitrogen). sgRNAs were quantified by Qubit RNA BR kit and diluted at 50 ng/μl and stored as single use aliquots. The efficiency of each sgRNA was assessed by injecting WT embryos with equal volume of previously diluted nls-Cas9 protein (IDT; 0.5 μl of nls-Cas9 added with 9.5 μl of 20 mM HEPES; 150 mM KCI, pH 7.5) and sgRNA, incubated at 37°C for 5 min and then 1 nl was injected in 1–2 cell stage embryos. At 24–72 hpf, 12–16 embryos from each sgRNA were individually collected and genomic DNA was extracted by heat shock denaturation in 50 mM NaOH (95°C for 20 min). For each embryo, PCR was performed on genomic DNA by using Q5 High-Fidelity Taq Polymerase (New England Biolabs) followed by T7 endonuclease I assay (New England Biolabs) to detect mutations. For T7 endonuclease I assay, 10 μl of PCR product was incubated with 0.5 μl of T7e1 enzyme (New England Biolabs) for 30 min at 37°C. Digested and undigested fragments were run in parallel in 2% agarose gel to assess the presence of indels. Efficiency was calculated as the number of embryos that show a positive result based on T7e1 assay divided by the total number of embryos assayed for each sgRNA.

All sgRNAs demonstrated to generate indel mutations were injected into the 1-cell embryos generated by an incross of uhrf1^−/+ adults as previously described. The resulting F₀ larvae were considered crispants. For each clutch and each sgRNA, uhrf1^−/− mutants were divided from phenotypically WT siblings at 5 dpf based on morphological differences and used for following analysis.