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.