CRISPR/Cas9 Editing of Zebrafish cdipt

A detailed procedure for CRISPR/Cas9 editing in zebrafish has been described previously [36 (link)]. Blast search of the zebrafish genome confirmed the existence of a single cdipt paralog in zebrafish (NM_207088). The cdipt target in this study was 5’- GGTTCACCAGCAAACACATGGTGG-3’ in exon 3. One-cell-stage AB WT embryos were injected with gRNA and Ca9 mRNA with a Picopump (World Precision Instruments). Potential founders (F₀) were outcrossed to AB WT fish. Genomic DNA was isolated from single F₁ embryos at 6 dpf and genotyped using high resolution melt (HRM) analysis. A cdipt sequence spanning the CRISPR/Cas9 target site was amplified with the following primers: F: 5’-AGCTGGAACAGAAAAGTGTAGGA-3’; and R: 5’-TAGGTACAAAATTTGGTGCAATG-3’. Carriers were identified and outcrossed ultimately to the F₃ generation. In-cross progeny from the F₃ and F4 generations were characterized in this study.

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Smith L., Fabian L., Al-Maawali A., Noche R.R, & Dowling J.J. (2020). De novo phosphoinositide synthesis in zebrafish is required for triad formation but not essential for myogenesis. PLoS ONE, 15(8), e0231364.

Publication 2020

Picopump

Cell Crispr Embryos Exon Fish Genomic Mrna Primers Zebrafish

Corresponding Organization : University of Toronto

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Variable analysis

independent variables

Injection of gRNA and Cas9 mRNA into one-cell-stage AB WT embryos

dependent variables

Genotype of F1 embryos at 6 dpf, as determined by high resolution melt (HRM) analysis of the cdipt sequence spanning the CRISPR/Cas9 target site

control variables

AB WT fish used as the genetic background for the experiments
Primers used for amplifying the cdipt sequence spanning the CRISPR/Cas9 target site

controls

Positive control: None specified
Negative control: None specified

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