gRNA and Cas9-encoding mRNA were co-injected into one-cell stage zebrafish embryos. Unless otherwise indicated, each embryo was injected with 2 nl of solution containing ~12.5ng/µl of gRNA and ~300ng/µl of Cas9 mRNA. On the next day, injected embryos were inspected under stereoscope and were classified as dead, deformed or normal phenotypes. Only embryos that developed normally were assayed for target site mutations using T7 Endonuclease I assay or DNA sequencing (see below). Genomic DNA was extracted from either single embryos or a pool of ten embryos as previously described21 (link).
Protocol detail
Cas9-mediated Zebrafish Genome Editing
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Corresponding Organization :
Other organizations : Massachusetts General Hospital, Center for Cancer Research
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Variable analysis
independent variables
- Amount of gRNA co-injected (12.5ng/µl)
- Amount of Cas9 mRNA co-injected (300ng/µl)
- Phenotypes of injected embryos (dead, deformed or normal)
- Target site mutations in normally developed embryos (assayed using T7 Endonuclease I assay or DNA sequencing)
- Volume of solution injected per embryo (2 nl)
- Extraction of genomic DNA from either single embryos or a pool of ten embryos
- Not explicitly mentioned
- Not explicitly mentioned
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