Experiment Validating Traffic Light Reporter 2 (TLR2) Mouse Model in Homozygous Blastocysts using SpCas9 Experiment Condition Guide 26-2 Assay Description: Reporter activation by NHEJ is calculated as the percent of blasts that express tagRFP out of the total blasts imaged. Approximately one third of NHEJ events are detected. Tissue Measured: Measurement Type: Editing Efficiency Record ID 15000005232 Editor Alt-R™ S.p. Cas9 Nuclease V3 Delivery System Delivery System Subtype Guide R26-2 Vector Model Species Mouse Model Name TLR-2 homozygous blastocyst Application Method Electroporation Application Site Dosage Injection Frequency Injection Rate Injection Volume Days post injection 96 hours Editor Format RNP Antidote Id Antidote Description Measured Values Samples Size: 0   Replicate Result (Editing Efficiency) Measurement Units: # blasts assessed by sequencing Mean # blasts analyzed  1 120 blasts  Measurement Units: # blasts assessed for RFP activation Mean # blasts analyzed  1 156 blasts  Measurement Units: % of blasts with RFP activation Mean 44.2  1 44.2  Measurement Units: % of blasts with edits Mean 53.33  1 53.33

Validation of Traffic Light Reporter 2 (TLR2) Mouse Model in Homozygote Blastocysts Zygotes were electroporated with Cas9 RNP along with guides and cultured 96 hours to blastocyst stage. Blastocysts were imaged for tagRFP fluorescence and scored for fluorescence signal.