CRISPR/Cas9 screen for genome-wide interrogation of essential MYC binding sites in cancer cells

Abstract

The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate expression of adjacent genes in a cell type- and developmental stage-specific manner. To date, a comprehensive analysis of direct MYC targets with essential roles in different types of cancer is missing. To enable identification of functional MYC binding sites and corresponding target genes, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. As a proof of principle, using this library we identified several known and novel MYC targets critical for K562 chronic myelogenous leukemia cells and uncovered specific features of essential E-boxes. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in any cancer type.

TransCRISPR - sgRNA design tool for CRISPR/Cas9 experiments targeting DNA sequence motifs

“…Lentiviral particles were produced in HEK293T cells using 2 nd generation packing plasmids and Calcium Phosphate Transfection Kit (Invitrogen, Carlsbad, CA, US). K562 cells were transduced as described previously 27 . Next day 3 µg/ml of puromycin was added and cells were selected for 96 h. After selection, cells were cultured in RPMI medium with 1 µg/ml of puromycin and collected for DNA and RNA isolation 7 days post transduction.…”

“…Apart from studying protein-coding genes, pooled CRISPR/Cas9 screens have been also utilized to characterize regulatory elements: promoters, enhancers, etc. Both nuclease Cas9 and dCas9 have been recently used to study transcription factors (TFs), their associations with tumorigenesis and identification of essential target genes (15, 16, 17, 18, 19, 20).…”