Figure 5.

(A) Structural domains and selective targeting of NRF2. The R34G mutation occurs in exon 2 of the NRF2 gene, which encodes the Neh2 Domain of the NRF2 protein, shown in upper panel. The lower panel presents a schematic of the creation of a new CRISPR/Cas9 PAM site through the R34G mutation (TCG → TGG). (B) Recreation of the R34G mutation in a NRF2 gene expression plasmid (pcDNA3-EGFP-C4-NRF2, Addgene). The NRF2 expression plasmid was mutated to contain the R34G mutation using CRISPR-directed mutagenesis with two CRISPR/Cas12a cleavage sites and a duplexed oligonucleotide. (C) Proof-of-concept in vitro cleavage reaction using the R34G mutation as a CRISPR/Cas9 cleavage site. The NRF2 expression plasmid was used to test the cleavage capacity of Cas9 with the new R34G mutation. Wildtype (WT NRF2) and mutated (R34G NRF2) NRF2 expression plasmids were amplified and purified amplicons were used for cleavage reactions. Reactions were visualized by gel electrophoresis. Lanes 1 and 5 are the NRF2 amplicons incubated with buffer only (negative control). Lanes 2 and 5 are NRF2 amplicons incubated with a nonspecific RNP (HBB RNP). Lanes 3 and 6 are NRF2 amplicons incubated with the R34G RNP. The red bars (right handside of ladder) indicate the size of uncut amplicons (901 bp) and cleavage products (222 & 679 bp).