Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis
(A) Crystal structure of DOT1L KMT domain binding with the EPZ-5676. The VVEL293MM mutation site was colored in yellow (PDB: 4HRA). (B) SDS-PAGE gel of purified recombinant DOT1L KMT domains of both wild-type and VVEL293MM mutant proteins. (C) Relative in vitro KMT activity of DOT1L wild-type and VVEL293MM mutant protein. The in vitro KMT activity was determined by a biochemical methyltransferase assay, which measured the methyltransfer level of radiolabeled SAM to purified nucleosomal substrates in the presence of a wild-type or mutant enzyme. To ensure that quantifications were made in the linear range of detection, the assay was performed at several DOT1L concentrations. The top panel shows a representative SDS-PAGE gel autoradiograph of the in vitro KMT assay with KMT proteins and concentrations of EPZ-5676 indicated. The lower panel plots the average integrated autoradiography intensity from two independent preparations of wild-type and mutant DOT1L KMT proteins. (D) Relative sensitivities of wild-type and VVEL293MM DOT1L to EPZ-5676. To normalize for the difference in basal activity between the wild-type and VVEL293MM DOT1L, the in vitro KMT assay reaction volume of the wild-type protein was increased three-fold. The top panel shows a representative SDS-PAGE gel autoradiograph of the in vitro KMT assay with KMT proteins and EPZ-5676 concentrations indicated. The lower panel plots the average results from three independent enzymatic preparations. All error bars shown represent s.e.m.