Experiment Validating Gene Editing Reporter 19 (GER19) Mouse Model in Heterozygous Blastocysts Experiment Condition Dual guides Assay Description: Reporter activation by NHEJ is calculated as the percent of blasts that express GFP out of the total blasts imaged. Tissue Measured: Measurement Type: Editing Efficiency Record ID 15000003449 Editor Alt-R® S.p. Cas9 Nuclease V3 Delivery System Delivery System Subtype Guide GER18/19 gRNA2 FWD; GER18/19 gRNA3 FWD Vector Model Species Mouse Model Name GER19 Heterozygous Blastocyst Application Method Electroporation Application Site Dosage Injection Frequency Injection Rate Injection Volume Days post injection 96 hours Editor Format ribonucleoprotein (RNP) Antidote Id Antidote Description Measured Values Samples Size: 0   Replicate Result (Editing Efficiency) Measurement Units: % of blasts with GFP activation Mean 76.34  1 81.25  2 71.43  Measurement Units: % of blasts with edits Mean 80.94  1 72.41  2 89.47  Measurement Units: Signal Mean # blasts analyzed  1 53 blasts analyzed for GFP  Mean # blasts analyzed  1 48 blasts analyzed for edits

Validation of Gene Editing Reporter 19 (GER19) Mouse Model in Heterozygous Blastocysts Zygotes were electroporated with Cas9 RNP along with test guides and were cultured 96 hours to blastocyst stage. Blastocysts were imaged for GFP fluorescence and scored for fluorescence signal.