Experiment Validating Traffic Light Reporter 7 (TLR7) Mouse Model for HDR in Heterozygous Blastocysts Experiment Condition RNP & ssODN Assay Description: Reporter activation by NHEJ is calculated as the percent of blasts that express Katushka2S (RFP) out of the total blasts imaged. Approximately one third of NHEJ events are detected. Tissue Measured: Measurement Type: Editing Efficiency Record ID 15000005027 Editor Alt-R® S.p. Cas9 Nuclease V3 Delivery System Delivery System Subtype Guide TLR7-g2-REV Vector Model Species Mouse Model Name TLR-7 Heterozygous Blastocyst Application Method electroporation Application Site Dosage Injection Frequency Injection Rate Injection Volume Days post injection 96 hours Editor Format RNP and ssODN repair template Antidote Id Antidote Description Measured Values Samples Size: 0   Replicate Result (Editing Efficiency) Measurement Units: # blasts assessed for HDR Mean # blasts analyzed  1 64 blasts  Measurement Units: # blasts assessed for RFP activation Mean # blasts analyzed  1 103 blasts  Measurement Units: % of blasts with RFP activation Mean 30.33  1 20  2 44  3 27  Measurement Units: % of blasts with Venus activation Mean 27.33  1 20  2 38  3 24

HDR Validation of Traffic Light Reporter 7 (TLR7) Mouse Model in Heterozygous Blastocysts Zygotes were electroporated with Cas9 RNP along with guides and HDR donor, and cultured 96 hours to blastocyst stage. Blastocysts were imaged for Venus and Katushka2S (RFP) fluorescence and scored for fluorescence signal.