Quantification of editing by NGS

On-target editing efficiency for Cas9/Cas12a nucleofected cells was measured by NGS. Amplicon sequencing libraries were prepared using a previously described rhAmpSeq amplification-based method.56 Briefly, the first round of PCR was performed using target-specific primers. A second round of PCR was used to incorporate P5 and P7 Illumina adapters to the ends of the amplicons for universal amplification. Libraries were purified using Agencourt AMPure XP system (Beckman Coulter, Brea, CA, USA) and quantified with qPCR before loading onto the Illumina MiSeq platform (Illumina, San Diego, CA, USA). Paired-end, 150 bp reads were sequenced using V2 chemistry. Data were demultiplexed using Picard tools v2.9 (https://github.com/broadinstitute/picard).