Pools of 30 to 50 embryos fertilized from in-crossing Tg(−8.3phox2bb:Kaede) adults were injected at the one-cell stage in the yolk with a solution containing 100 picogram (pg) of gene specific sgRNA, 2 μM Cas9 NLS nuclease (Synthego) and Phenol red. A total of eight injected F0 larvae were dissociated, used in T7 endonuclease I activity assays (NEB E3321) as previously described [16 (link)], and the percentage of indels was determined. PCR pair of primers per gene used for the T7E1 activity (S1 Table ) targeted the specific sgRNA region for each gene and amplified regions between 200 to 300 bp. Experimental replicates were done at least 3 times per gene for the injections and the genotyping. Live embryos at 48 hpf were genotyped using the Zebrafish Embryonic Genotyper (ZEG) as instructed by the manufacturer (InVivo Biosystems).
T7 endonuclease 1 activity assays
Manufactured by New England Biolabs
T7 endonuclease I is an enzyme that cleaves DNA at sites of non-complementary base pairing, such as those present in DNA heteroduplexes formed by hybridization of similar but not identical DNA sequences. The T7 endonuclease I activity assays provide a method to measure the activity of this enzyme.
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2 protocols using t7 endonuclease 1 activity assays
1
CRISPR Mutagenesis in Zebrafish Embryos
2
Genome Editing in Zebrafish Embryos
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Pools of 30 to 50 embryos fertilized from in-crossing Tg(−8.3phox2bb:Kaede) adults were injected at the one-cell stage in the yolk with a solution containing 100 picogram (pg) of gene specific sgRNA, 2 µM Cas9 NLS nuclease (Synthego) and Phenol red. A total of eight injected F0 larvae were dissociated, used in T7 endonuclease I activity assays (NEB E3321) as previously described (Baker et al., 2022 (link)), and the percentage of indels was determined. PCR pair of primers per gene used for theT7E1 activity (Supplementary table 1 ) targeted the specific sgRNA region for each gene and amplified regions between 200 to 300 bp. Experimental replicates were done at least 3 times per gene for the injections and the genotyping.
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