Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis
(A) Schematic of the vector used to derive clonal MLL-AF9; NrasG12D leukemia RN2c cells that express a human codon-optimized S. pyogenes Cas9 (hCas9) and the vectors used for lentiviral sgRNA transduction. A GFP reporter was used where indicated to track sgRNA positive selection. LTR: long terminal repeat promoter, PGK: phosphoglycerate kinase 1 promoter, Puro: puromycin resistance gene, U6: a Pol III-driven promoter, sgRNA: chimeric single guide RNA, EFS: EF1α promoter, GFP: green fluorescent protein. (B) Schematic of DOT1L protein domain. A library of 73 sgRNAs was designed targeting the KMT domain of DOT1L. The DOT1L inhibitor EPZ-5676 targets the KMT domain. KMT, lysine methyltransferase. (C) Experimental workflow of domain-focused CRISPR indel mutagenesis screening. A pooled sgRNA library targeting the DOT1L KMT domain was introduced to RN2c via lentiviral transduction, followed by continuous exposure to EPZ-5676. Genomic DNA of indicated cell populations was isolated and harvested for sgRNA cassette quantification and sgRNA-induced indel identification by deep sequencing. (D) CRISPR indel mutagenesis genetic screening of DOT1L identified EPZ-5676 resistant mutants. The RN2c cells were treated with EPZ-5676 at day 3 post-transduction, in which a fraction of the cells was harvested and used as a reference time point (day 0). Cells were initially treated with 100 nM of EPZ-5676 at day 0. The inhibitor dosage was increased to 500 nM at day 20. At day 41, an EPZ-5676 resistant GFP+/sgRNA+ population was identified, harvested, and saved for subsequent deep sequencing experiments. (E) Quantitative reverse transcription PCR (qRT–PCR) of relative mRNA expression levels in parental and EPZ-5676 resistant RN2c cell lines after a 6-day treatment with indicated the concentration of EPZ-5676 or DMSO. Results were normalized to Gapdh, with the relative mRNA level in the DMSO-treated cells set to 1 (n = 3). (F) Pie charts of the relative abundance for 73 individual sgRNAs at indicated time points. Each slice represents the abundance of a single sgRNA. At day 0 (left), all 73 sgRNAs targeting the DOT1L KMT domain were detected at roughly equal abundance. At day 41 (right), DOT1L sgRNA #47 dominated the population, representing >95% of the GFP-positive cells. (G) A proliferation competition assay of RN2c cells transduced with indicated sgRNAs under the treatment of EPZ-5676 or DMSO shows that DOT1L sgRNA #47 induced mutant(s) confer resistance to EPZ-5676. An sgRNA targeting a Rosa locus and a random sgRNA targeting the DOT1L KMT domain (DOT1L sgRNA #64) served as negative controls. (H) Nucleotide and protein sequence diagram of the wild-type and DOT1L mutation induced by sgRNA #47 CRISPR mutagenesis under the treatment of EPZ-5676. Indel mutations were observed flanking the sgRNA cut site in exon 8 of DOT1L. The indel mutation corresponded to a VVEL to MM mutation at residue 293. (I) The effect of ectopic overexpression of DOT1L wild-type or mutant on RN2c cell proliferation under the treatment of EPZ-5676. RN2c cells were retrovirally transduced with wild-type or mutant human DOT1L cDNA. After a 9-day drug treatment, the relative cell accumulation corresponding to each indicated EPZ-5676 concentrations was measured and normalized to the DMSO treated cells (n = 3). All error bars shown represent s.e.m.