title: Amplicon seq of off-target OT2- mouse - GD - Rep2
design: Putative CRISPR off-target sites for the gRNA ACGGGATGCCGGGACTTAAG were identified in the mouse genome (mm10) using the online tool CRISPOR v5.2 (http://crispor.gi.ucsc.edu/crispor.py). 14 sites with Cutting Frequency Determination (CFD) >0.1 were analyzed by amplicon sequencing. Mouse N2a cells were infected with engineered Herpes simplex virus 1 (HSV-1) expressing Cas9 and gRNA ACGGGATGCCGGGACTTAAG (virus GD, sample 5-8), or with control virus expressing Cas9 and an unspecific gRNA (virus GD-ns, sample 1-4). As a control, we used uninfected N2a cells (samples 9-12). N2a cells were infected with GD and GD-ns at MOI=1 and cells collected after 3 days. DNA was extracted using Qiagen DNeasy kit. PCR amplicons, ranging from 180 to 300bp for the 14 sites, were amplified using Phusion Plus high-fidelity polymerase (ThermoFisher, USA) on a Biorad Thermocycler. Amplicon were barcoded and sequenced on an Illumina Nextseq 2000 using 2x150 read format.