Computational protocol: nr3c1 null mutant zebrafish are viable and reveal DNA-binding-independent activities of the glucocorticoid receptor

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Protocol publication

[…] Gr mutant cells were generated using the CRISPR/Cas9-mediated genome editing. Gene-specific guide RNA (sgRNA) was designed against an optimal CRISPR site in exon 2 of gr (NM EF567112.1) using the CHOPCHOP software (available at: Gr target sequence and CRISPR oligonucleotides are listed in Table . BLAST analysis of the target sequence revealed no specific binding with other genes. sgRNA was generated according to previously described methods and in vitro transcribed using MEGAshortscript T7 kit (Life Technologies, AM1354). Cas9 mRNA was transcribed from linearized pCS2-nls-zCas9-nls plasmid (Addgene, Plasmid #47929) using mMessage Machine SP6 kit (Life Technologies, AM1340).Fertilized eggs were injected with 1 nl of a solution containing 400 ng/μl Cas9 mRNA and 50 ng/μl gRNA. Genomic DNA was extracted from 5-dpf larvae from individually injected eggs to verify the presence of mutations and confirm the activity of the guide RNA. Injected embryos were raised to adulthood and F0 founders selected by genotyping (see below). Embryos collected from the outcrosses between these F0 founders and WT were raised and genotyped to confirm germline transmission of the mutation (F1 generation). Heterozygous mutants with the same mutation were selected and crossed, to obtain homozygous mutant embryos (F2 generation). [...] Larvae or adult fish were anesthetized in tricaine and a small fragment of the caudal fin was cut with a sharp blade. Genomic DNA was extracted using the HotSHOT protocol.Mutations in F0 were detected using heteroduplex mobility assay (HMA). In this case, genomic fragments at the target sites were amplified by PCR with 5x HOT FIREPol® Blend Master Mix (Solis BioDyne, 04-25-00125) and the locus-specific primers (gr-F1 and gr-R1) listed in Table .PCR conditions were as follows: 15 min at 95 °C, 35 cycles at 95 °C for 20 s, 60 °C for 30 s and 72 °C for 30 s. The resultant PCR amplicons were electrophoresed on a 15% polyacrylamide gel (Life Technologies, NP0323PK2). For verification, PCR products from fish harbouring indel mutations were subjected to sequencing. Poly Peak Parser software ( was used for identification and sequence characterization of heterozygous mutant carriers generated by genome editing.Screening primers for heterozygous and homozygous fish, gr-F2 and gr-R2 (see Table  and Supplemental Fig. , panel C), were designed to amplify a 82-bp region across the gr sgRNA target region. PCR products were resolved with ethidium bromide-stained 3% agarose low EEO gel (Fisher BioReagents, BP160-500) in order to identify gr +/+, gr +/− and gr −/− samples. gr s357/s357 mutants were identified by sequencing of a 223-bp fragment which includes the gr s357 point mutation obtained by genomic DNA PCR amplification with primers s357-F1 and s357-R1 (Table ). […]

Pipeline specifications

Software tools CHOPCHOP, Poly Peak Parser
Databases Addgene
Application Genome edition
Organisms Danio rerio, Caenorhabditis elegans, Mus musculus
Diseases Intestinal Diseases
Chemicals Hydrocortisone