Off target analysis of CRISPR-Cas9 edited H99 callus in maize

To identify potential off-target mutations generated by the NRT1.1 genome editing system, Illumina deep sequencing on T0 seedling leaf tissue from event 1 was performed. This sequencing run produced 70 million total paired reads which gives an average read depth of 8.75X across the maize genome. These sequence reads were aligned to Oh43, which is the most closely related inbred to H99 with a complete genome assembly. Each of the 20 genomic regions predicted as off target sites for the three NRT1.1-gRNAs were visually inspected for sequence variation indicative of editing. The average read depth within these off-target regions was 13.6. When allowing up to three sequence mismatches, only one showed evidence of off-target mutagenesis, which coincided with NRT1.1D, the 4th member of the NRT1.1 gene family.为鉴定NRT1.1基因组编辑系统所产生的潜在脱靶突变，本研究对事件1的T0代幼苗叶片组织开展了Illumina深度测序（Illumina deep sequencing）。本次测序共产出总计7000万条双端读段（paired reads），在玉米全基因组范围内的平均测序深度为8.75倍。将所得序列读段比对至Oh43参考基因组——Oh43是与H99亲缘关系最近且具备完整基因组组装结果的近交系。针对3条NRT1.1向导RNA（gRNA）所预测的20个脱靶靶位点所在的基因组区域，我们逐一进行了可视化检视，以查找指示编辑发生的序列变异。这些脱靶区域内的平均测序深度为13.6倍。当允许最多3个序列错配时，仅1个区域存在脱靶诱变的证据，该区域与NRT1.1基因家族的第4个成员NRT1.1D相契合。