Figure 2

In vitro cleavage assay with plasmids containing sequences with PAM mutations. (a) Top: schematic of the plasmids containing sequences with wild-type and oncogenic mutations. PCR amplicons of relevant wild-type proto-oncogene cDNAs were subcloned into the commercial T-blunt cloning vector (T-Blunt PCR cloning Kit, SolGent, Seoul, South Korea) using the manufacturer’s protocol. Single-base-pair-substituted mutations (KRAS: c.35G>A, c.35G>T, c.34G>T, c.35G>C, c.34G>C, GNAQ: c.626A>T) were constructed with site-directed mutagenesis using appropriate primer sets (Supplementary Table 3). Plasmids can be linearized by the restriction enzyme NcoI. Bottom: the sequences of wild-type and recurrent KRAS mutations in the COSMIC database. The PAM sequence (5′-TGG-3′) for SpCas9 is underlined in blue. Missense mutations are shown in red. (b) In vitro cleavage assay using SpCas9 with linearized plasmids containing wild-type and mutant KRAS sequences. Target plasmids were first linearized with the restriction enzyme NcoI (New England Biolabs, Ipswich, MA, USA) for 37 °C for 1 h (10 μl reaction in NEB buffer 3.1). The linearized product was further cleaved by treatment with a wild-type sequence-specific CRISPR nuclease (Cas9 100 ng, sgRNA 70 ng, 10 μl reaction in NEB buffer 3.1 at 37 °C, 1 h). CF, cleaved fragment; LF, linearized fragment. (c) The sequences of wild-type and recurrent GNAQ mutation in the COSMIC database. The PAM sequence (5′-TTG-3′) for FnCpf1 is underlined in blue. (d) In vitro cleavage assay using FnCpf1 with linearized plasmids containing wild-type and mutant GNAQ sequences.