NLB18 Maize was generated using CRISPR-Cas editing by biolistic transformation with seven plasmids.

Northern Leaf Blight (NLB) is induced by the fungal pathogen Exserohilum turcicum (previously called Helminthosporium turcicum).

In NLB18 Maize plants, the NLB sensitive allele of the nlb18 gene was replaced with a resistance allelic version (Cs-nlb18 (res)_MAIZE) from the same locus, resulting in improved resistance to NLB.

Two guide RNAs were used to excise the nlb18 sensitive allele. The first gRNA was targeting a sequence in the nlb18 gene promoter, the second gRNA was targeting its 3’UTR. Simultaneously a repair template DNA encoding the resistant allele nlb18 (res) was supplied to repair the targeted double strand break.

The intended replacement of the disease sensitive allele was characterized by next generation sequencing. Developers used Southern-by-Sequencing analysis and breeding processes to select the final plant line, which contains the disease resistant allele nlb18 (res) at its native genomic locus and does not contain any unintended plasmid DNA sequence.

Sources:

1. Patent WO2018071362A1
2. USDA-APHIS air inquiry 17-076-01