Experiment Independent validation of Sontheimer delivery platform using heavily modified guide RNAs complexed with Cas9 proteins to deliver CRISPR/Cas9 to mouse brain Experiment Condition DNMT1 C20/T2 SpyCas9 Males Assay Description: Editing efficiency calculated by counting eGFP expressing cells (DAB+) 0.5 mm from either side of the injection track compared to hematoxylin labeled nuclei in mouse striatum. Cell counting is done with FIJI macro. #DAB+ (eGFP+) cells/total number of cells in ROI *100 = Percent edited cells in ROI Tissue Measured: striatum Measurement Type: Editing Efficiency Record ID 15000003052 Editor SpyCas9-3xNLS Delivery System Delivery System Subtype Guide STS204 (DNMT1; Sp_t2:Sp_c20_Dnmt1) Vector Model Species Mouse Model Name B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J Sample Age 6-8 weeks Sample Sex Male Application Method stereotactic bilateral intra-striatum injection, coordinates AP+ 1.0 mm relative to bregma, ML +/- 2.0 mm, DV -3.0 mm Application Site Striatum- brain Dosage 100 uM Injection Frequency once Injection Rate 500 nL/min Injection Volume 2 microliters left hemisphere, 2 microliters right hemisphere Days post injection 14 days Editor Format ribonucleoprotein (RNP) Antidote Id Antidote Description Antibody RRID RRID:AB_2536526 Antibody Other ID Thermo Fisher Scientific Cat# G10362 Antibody Description GFP Recombinant Rabbit Monoclonal Antibody, Thermo Fisher Scientific #G10362 Antibody RRID RRID:AB_2313606 Antibody Other ID Vector Labs Cat# BA-1000 Antibody Description Vector Labs Goat Anti-Rabbit IgG Biotinylated Cat. #BA-1000 Measured Values Samples Size: 3   Replicate Result (Editing Efficiency) Measurement Units: % edited cells in ROI Mean 0.28  1 0.26  2 0.09  3 0.48