Mutations in the EGFR kinase are a cause of non-small-cell lung cancer. Deletions in exon 19 and the L858R (2573T>G) single point mutation constitute about 90% of all EGFR mutations. Selectively inactivate only mutant, not normal allele, could benefit patients with such mutations. Here, the editing efficacy and selectivity of CRISPR-Cpf1 and -Cas9 systems on EGFR L858R mutant allele were analyzed by dual-reporter assay in vitro. Near the mutation site, there are two TTTN protospacer adjacent motifs (PAMs) for Cpf1. 2573T>G substitution also leads to occurrence of a novel NGG PAM for Cas9. Thus we designed two AsCpf1 gRNA (gRNA1 and gRNA2) and one SpCas9 gRNA (gRNA3) and evaluated their potency and allele specificity in vitro using a dual fluorescent protein-based bioassay system. As a result, both AsCpf1 and SpCas9 demonstrated robust activities to induce specific editing of only 2573T>G mutant EGFR, not wild-type sequence. Our results support the potential applicability of both Cpf1 and Cas9 in precision medicine through highly specific disruption of mutant oncogenes.
