Experiment Validating Traffic Light Reporter 7 (TLR7) Mouse Model for NHEJ in Heterozygous Blastocysts Experiment Condition RNP 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 15000005026 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 ribonucleoprotein (RNP) Antidote Id Antidote Description Measured Values Samples Size: 0   Replicate Result (Editing Efficiency) Measurement Units: # blasts assessed by sequencing Mean # blasts analyzed  1 117 blasts  Measurement Units: # blasts assessed for RFP activation Mean # blasts analyzed  1 169 blasts  Measurement Units: % of blasts with RFP activation Mean 35.67  1 34.29  2 50  3 28.57  4 30.88  5 34.62  Measurement Units: % of blasts with edits Mean 96.08  1 100  2 100  3 84.31  4 100

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