CRISPR-Cas9 Knockout of Meis1 and Pbx3

sgRNAs were designed targeting meis1 and pbx3 using CRISPRSCAN (https://www.crisprscan.org/?page=gene) (options: organism = ‘Frog-Xenopus tropicalis’, Gene = ‘pbx3’ or ‘meis1’, Cas9-NGG, In Vitro T7 promoter). PCR was performed as described in Bhattacharya et al. (2015) (link). SgRNA was transcribed using T7 mMachine kit (Ambion). Guides were injected into 2 blastomeres of 4 cell embryos with 1.5 ng Cas9 (Bhattacharya et al., 2015 (link); Nakayama et al., 2013 (link)). All guides were injected at a dose of 400 pg/embryo.

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Kakebeen A.D., Chitsazan A.D., Williams M.C., Saunders L.M, & Wills A.E. (2020). Chromatin accessibility dynamics and single cell RNA-Seq reveal new regulators of regeneration in neural progenitors. eLife, 9, e52648.

Publication 2020

T7 mMachine kit

Blastomeres Cell Embryo Frog Gene Pbx3 Xenopus

Corresponding Organization :

Other organizations : Seattle University, University of Washington

Top 5 similar protocols

Variable analysis

independent variables

SgRNA targeting meis1
SgRNA targeting pbx3

dependent variables

Phenotypic outcomes of CRISPR/Cas9-mediated gene editing

control variables

Organism (Frog-Xenopus tropicalis)
Cas9 protein concentration (1.5 ng per embryo)
SgRNA concentration (400 pg per embryo)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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