Comprehensive Genome Editing Evaluation

For Sanger sequencing, target sites were PCR amplified (Table S1) using PrimeStar GXL polymerase (Takara), and PCR cleanup was done using ExoSap-IT Express (Thermo Fisher). Chromatograms were analyzed using EditR to determine base editing efficiencies.⁴⁹ (link) Candidate OTSs were identified with CRISPOR, and the top five sites, by cutting frequency determination (CFD) score, for which PCR products were successfully obtained were selected.⁵⁰ (link)^,51 (link) Target sites were PCR amplified (Table S1) using PrimeStar GXL polymerase (Takara), and a second round of PCR was used to add Illumina flow cell binding sequences and barcodes. PCR products were purified with AMPure XP beads (Beckman Coulter), analyzed for integrity on a 2200 TapeStation system (Agilent), and quantified by Qubit dsDNA high-sensitivity assay (Invitrogen) before pooling and loading onto an Illumina MiSeq. Following demultiplexing, resulting reads were analyzed with CRISPResso2 for editing frequency.⁵² (link) To analyze the number of AAV integration events at the on-target site, we followed a previously established method.⁵³ (link) Sequencing files were aligned to AAV vector sequences using the bwa program (version 0.7.17) and sorted with samtools (version 1.6), and the number of reads that had vector sequences were counted and normalized to number of reads that mapped to the target amplicon.