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Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis

Fig 3

Domain-focused CRISPR genetic screening identifies an EZH2 mutant allele that is resistant to the drug EPZ-6438.

(A) Schematic of EZH2 protein domain. A library of 66 sgRNAs was designed targeting the KMT domain of EZH2. The EZH2 inhibitor EPZ-6438 inhibits the KMT function. (B) CRISPR indel mutagenesis genetic screening identified EZH2 mutants resistant to EPZ-6438 treatment. RN2c cells were treated with 5 uM EPZ-6438 at day 3 post-transduction, at which point a fraction of the cells was harvested and used as a reference time point (day 0). At day 42, an EPZ-6438 resistant sgRNA+/GFP+ population was identified, harvested, and saved for subsequent deep sequencing experiments. (C) Pie charts of the relative abundance for individual 66 sgRNAs at indicated time points. Each slice represents the abundance of a single sgRNA. At day 0 (left), all 66 sgRNAs targeting the EZH2 KMT domain were detected at roughly equal abundance. At day 42 (right), EZH2 sgRNA #52 dominated the population, representing >95% of the GFP+ cells. (D) Nucleotide and protein sequence diagram of the wild-type and EZH2 mutation induced by sgRNA #52 CRISPR mutagenesis under the selection pressure of EPZ-6438. The indel mutations were observed in the exon 17 of EZH2 flanking the sgRNA cut site. Indel mutations were observed in exon 17 of EZH2 flanking the sgRNA cut site. The indel mutation corresponded to a TR to KK mutation from residue 678. (E) The effect of ectopic overexpression of EZH2 wild-type or TR683KK mutant on RN2c cell proliferation under the treatment of EPZ-6438. The RN2c cells were retrovirally transduced with wild-type or mutant human EZH2 cDNA. After a 5-day drug treatment, the relative cell accumulation corresponding to each indicated EPZ-6438 dosage was measured and normalized to the DMSO treated cells (n = 3). (F) qRT–PCR of relative mRNA expression levels in the indicated cell lines after a 5-day treatment with EPZ-5676 or DMSO. Results were normalized to Gapdh, with the relative mRNA level in the DMSO-treated cells set to 1 (n = 3). All error bars shown represent s.e.m.

Fig 3