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Experiment: Validating Gene Editing Reporter 14 (GER14) Mouse Model in Heterozygous Blastocysts

PI:   Stephen A Murray, PhD 

Description: WT female mouse and homozygous male mouse containing the Gene Editing Reporter 14 (GER14) reporter at the Rosa26 locus were mated. Females were super-ovulated and zygotes harvested. Zyotes were electroporated with various CRISPR/Cas9 combinations (or Cre) and were cultured 96 hours to blastocyst stage. Blastocysts were imaged for Katushka2S (RFP) fluorescence and scored for fluorescence signal. Blastocysts were then PCR amplified for the reporter allele and Sanger sequenced. ICE analysis tool from Synthego was used to score edits. NOTE: Some blasts did not PCR amplify well and it could not be determined if they had edits. Total blasts analyzed expressing the fluorescent reporter, 46-164. Total blast analyzed by Sanger sequencing, 39-142.
Editing Assay:
  • Reporter activation by NHEJ is calculated as the percent of blasts that express Katushka2S (RFP) out of the total blasts imaged.
  • Reporter editing efficiency assessed using PCR amplification of the reporter locus in individual blasts followed by Sanger sequencing. Percent edited blasts are expressed as the number of blasts with DNA changes in the target locus divided by the total number of blasts with a scorable chromatogram.
Download: Submitted files
Filter Results ..
  Editors
 Alt-R A.s. Cas12a (Cpf1) V3
 Alt-RĀ® S.p. Cas9 Nuclease V3
 CleanCapĀ® Cre mRNA
 EnGenĀ®Ā Sau Cas9
Guide Target Locus
 GER14 (CAG-Venus*,-Katushka2S,-akaLuc)
  Guides
 GER14-g1-Cpf1-FWD
 GER14-g1-SaC9-FWD
 GER14-g1-SpC9-FWD
Models
 GER14 Heterozygous Blastocyst
Select experimental variable to highlight records on the chart:

Note: Hover over the bars to view additional information

Results
Validating_Gene_Editing_Reporter_14_(GER14)_Mouse_Model_in_Heterozygous_Blastocysts
Editing Efficiency
Record Id Condition Editor Model Target Locus Guide Editing Efficiency Signal Editing Efficiency % of blasts with RFP activation Editing Efficiency % of blasts with edits Image
15000003591 GER14-g1-SpC9-FWD Alt-RĀ® S.p. Cas9 Nuclease V3 GER14 Heterozygous Blastocyst GER14 (CAG-Venus*,-Katushka2S,-akaLuc) GER14-g1-SpC9-FWD
15000003592 GER14-g1-SaC9-FWD EnGenĀ®Ā Sau Cas9 GER14 Heterozygous Blastocyst GER14 (CAG-Venus*,-Katushka2S,-akaLuc) GER14-g1-SaC9-FWD
15000003593 Cre CleanCapĀ® Cre mRNA GER14 Heterozygous Blastocyst
15000003594 GER14-g1-Cpf1-FWD Alt-R A.s. Cas12a (Cpf1) V3 GER14 Heterozygous Blastocyst GER14 (CAG-Venus*,-Katushka2S,-akaLuc) GER14-g1-Cpf1-FWD
https://stage.scge.mcw.edu/toolkit/data/experiments/experiment/18000000075/record/15000003591/

Zygotes were electroporated with SpCas9 RNP along with guides and cultured 96 hours to blastocyst stage. Blastocysts were imaged for Katushka2S (RFP) fluorescence and scored for fluorescence signal.
SaCas9 Validation of Gene Editing Reporter 14 (GER14) Mouse Model in Heterozygous Blastocysts

Zygotes were electroporated with SaCas9 RNP along with guides and cultured 96 hours to blastocyst stage. Blastocysts were imaged for Katushka2S (RFP) fluorescence and scored for fluorescence signal.
Cpf1 Validation of Gene Editing Reporter 14 (GER14) Mouse Model in Heterozygous Blastocysts

Zygotes were electroporated with Cpf1 RNP along with guides and cultured 96 hours to blastocyst stage. Blastocysts were imaged for Katushka2S (RFP) fluorescence and scored for fluorescence signal.
Cre Validation of Gene Editing Reporter 14 (GER14) Mouse Model in Heterozygous Blastocysts

Zygotes were electroporated with Cre mRNA and cultured 96 hours to blastocyst stage. Blastocysts were imaged for Katushka2S (RFP) fluorescence and scored for fluorescence signal.