Experiment Delivery of RNP containing chemically modified crRNA C20 with chemically modified tracrRNA T2-PS to determine the RNP distribution in TLR-MCV mouse brain Experiment Condition Sp_c20_MCV1a/Sp_t2_5-3_PS-tail Assay Description: Delivery and uptake testing of C20-Cy3/T2-PS (red fluorescence) and SpyCas9 by immunohistochemical staining (green fluorescecnce) after intrastriatal injections Tissue Measured: brain Measurement Type: Delivery Efficiency Record ID 15000000703 Editor SpyCas9-3xNLS Delivery System Delivery System Subtype Guide STS118 (TLR-MCV1a; Sp_t2-PS:Sp_c20_TLR_MCV1a) Vector Model Species Mouse Model Name TLR-MCV1 Sample Age 7-8 weeks Application Method Intra-Striatum Application Site brain Dosage ~150 µM RNP Injection Frequency once Injection Rate 500 nl/min Injection Volume 2 microliters Days post injection 24 hours Editor Format ribonucleoprotein (RNP) Antidote Id Antidote Description Antibody RRID RRID:AB_2798820 Antibody Other ID AB_2798820 Antibody Description Cas9 (S. pyogenes) (E7M1H) XP® Rabbit mAb antibody, Cell Signaling Technology Measured Values Samples Size: 2   Replicate Result (Delivery Efficiency) Measurement Units: Signal Mean 1 C20-Cy3 present and SpyCas9 present and 1 C20-Cy3 absent   1 C20-Cy3 present and SpyCas9 present  2 C20-Cy3 absent

Signals from Spycas9 and crRNA-Cy3 are present