Zebrafish CRISPR Genome Editing Protocol

For the initial screen, zebrafish TLAB strain zygotes were collected and injected through the chorion with a mix of 25 pg sgRNA, 300 pg Cas9 mRNA, and phenol red dye in a single mix. Embryos were grown to 24–30hpf and genomic DNA extracted from pools of 8–10 embryos (unless otherwise indicated) using the HotSHOT method [18] . For comparison between Cas9 mRNA and protein, higher levels of sgRNA were co-injected (200–300 pg). Cas9/sgRNA complex was formed by incubating protein with sgRNA at room temperature for 5 minutes before injection.

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Gagnon J.A., Valen E., Thyme S.B., Huang P., Ahkmetova L., Pauli A., Montague T.G., Zimmerman S., Richter C, & Schier A.F. (2014). Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs. PLoS ONE, 9(5), e98186.

Publication 2014

Chorion Embryos Genomic Mrna Protein Strain Zebrafish Zygotes

Corresponding Organization : Harvard Stem Cell Institute

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Variable analysis

independent variables

Injection of a mix of 25 pg sgRNA, 300 pg Cas9 mRNA, and phenol red dye
Co-injection of higher levels of sgRNA (200-300 pg) with Cas9 mRNA or protein
Formation of Cas9/sgRNA complex by incubating protein with sgRNA at room temperature for 5 minutes before injection

dependent variables

Not explicitly mentioned

control variables

Zebrafish TLAB strain zygotes
Growth of embryos to 24-30 hpf
Genomic DNA extraction from pools of 8-10 embryos (unless otherwise indicated) using the HotSHOT method

controls

No positive or negative controls were explicitly mentioned in the provided information.

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