Amplicon sequencing (AmpSeq) for on- and off-target analysis and NGS analysis

To assess the editing efficiency or to evaluate predicted off-targets, a ~200–240 bp fragment flanking the genomic target site was amplified by PCR using Q5 High-Fidelity polymerase (2XMM Master Mix (NEB), 1 µL crude cell lysate or 50 ng purified genomic DNA, and barcoded primers (see Supplementary Table ³ for locus-specific primers). The cycling program was performed according to the manufacturer’s instructions with an annealing temperature of 66 °C and an extension time of 15 s. All PCR reactions were pooled into a library and purified with magnetic beads (AMPure XP beads, Beckman Coulter). Bead purification was performed by applying the following reaction steps. Two micrograms of the pooled amplicons were end-repaired and dA-tailed (NEBNext Ultra II End Repair/dA-tailing Module, NEB) and used for ligation of Illumina indices (Blunt/TA Ligase master Mix, NEB). The concentration was measured using the dsDNA HS Kit (Thermo Fisher Scientific) on a Qubit spectrophotometer and diluted to 4 nM. The library was denatured with 0.2 M NaOH for 5 min at RT and diluted to 10 pM using the buffer provided in the MiSeq 300PE v2 Kit. The sample was sequenced with 12.5 pmol PhiX on a MiSeq Instrument (Illumina). For de-multiplexing an in-house python script was used and individual samples were analyzed using CRISPResso V1.0.13⁵⁷ to extract % NHEJ. A threshold of >20,000 reads was chosen.