Experiment Independent validation of Gong delivery platform using RNP-loaded nanocages to deliver CRISPR/Cas9 to mouse brain Experiment Condition NC-RVG Females Assay Description: On-target editing efficiency was calculated by counting the number of edited Neurons (NeuN+/GFP+ and tdTomato+/RFP+) out of the total number of neurons (NeuN+/GFP+) in a defined region of interest in the brain, expressed as a percentage of neurons in the region of interest (ROI). Off-target editing analysis: peripheral tissues were fixed, cryosectioned and imaged directly for RFP (tdTomato). Presence or absence of signal above background was recorded for each tissue assessed. Biological replicates by condition were as follows: NC-CPP Males: On-target N=3, Off-target N=1; NC-CPP Females: On-target N=3, Off-target N=1; NC-RVG Males: On-target N=3, Off-target N=1; NC-RVG Females: On-target N=3, Off-target N=2; NC No Ligand Male: On-target N=3, Off-target N=2; NC No Ligand Female: On-target N=3, Off-target N=1. *Inconclusive: Signal in cells surrounding epididymal duct/testis germinal epithelium was observed in one animal (1 out of 3-4 tissue sections) Tissue Measured: eye Measurement Type: Editing Efficiency Record ID 15000003346 Editor sNLS-SpCas9-sNLS Delivery System RNP-NC-RVG Delivery System Subtype Nanoparticle Guide Ai14 gRNA Vector Model Species Mouse Model Name B6.Cg-Gt(ROSA)26Sor^tm14(CAG-tdTomato)Hze/J Application Method stereotactic bilateral intra-striatal injection, coordinates: Ant: + 1.2 mm from bregma and M/L +/-1.6 mm from midline; Depth = 3.25/3.75 mm Application Site brain (striatum) Dosage Injection Frequency once Injection Rate 0.2 ul per minute Injection Volume 1 microliter per hemisphere Days post injection 14 days Editor Format Nanocage with CRISPR/Cas9-RNP Antidote Id Antidote Description Antibody RRID RRID:AB_2209751 Antibody Other ID Antibody Description Antibody RRID RRID:AB_2532994 Antibody Other ID Antibody Description Antibody RRID RRID:AB_10711040 Antibody Other ID Antibody Description Antibody RRID RRID:AB_141637 Antibody Other ID Antibody Description Antibody RRID RRID:AB_2813835 Antibody Other ID Antibody Description Antibody RRID RRID:AB_141607 Antibody Other ID Antibody Description Measured Values Samples Size: 0   Replicate Result (Editing Efficiency) Measurement Units: Signal Mean 0 of 2 present  1 absent  2 absent