design: For rice protoplasts, genomic DNA was then extracted with the CTAB method. For tomato protoplasts, Phire Plant Direct PCR Master Mix (ThermoFisher) was used for direct PCR amplification. Targeted mutagenesis was quantified by next-generation sequencing (NGS) of PCR amplicons using barcoded primers. The PCR amplicons were sequenced by using an Illumina HiseqX platform.