EPIKOL, a chromatin-focused CRISPR/Cas9-based screening platform, to identify cancer-specific epigenetic vulnerabilities

Abstract

Dysregulation of the epigenome due to alterations in chromatin modifier proteins commonly contribute to malignant transformation. To discover new drug targets for more targeted and personalized therapies, functional interrogation of epigenetic modifiers is essential. We therefore generated an epigenome-wide CRISPR-Cas9 knock-out library (EPIKOL) that targets a wide-range of epigenetic modifiers and their cofactors. We conducted eight screens in two different cancer types and showed that EPIKOL performs with high efficiency in terms of sgRNA distribution, depletion of essential genes and steady behaviors of non-targeting sgRNAs. From this, we discovered novel epigenetic modifiers besides previously known ones that regulate triple-negative breast cancer and prostate cancer cell fitness. With further validation assays, we confirmed the growth-regulatory function of individual candidates, including SS18L2 and members of the NSL complex (KANSL2, KANSL3, KAT8) in triple negative breast cancer cells. Overall, we show that EPIKOL, a focused sgRNA library targeting approximately 800 genes, can reveal epigenetic modifiers that are essential for cancer cell fitness and serve as a tool to offer novel anti-cancer targets. With its thoroughly generated epigenome-wide gene list, and the relatively high number of sgRNAs per gene, EPIKOL offers a great advantage to study functional roles of epigenetic modifiers in a wide variety of research applications, such as screens on primary cells, patient-derived xenografts as well as in vivo models.

Genome-wide CRISPR screen identifies PRC2 and KMT2D-COMPASS as regulators of distinct EMT trajectories that contribute differentially to metastasis

“…Based on these initial results, we proceeded to perform a more focused CRISPR screen employing an sgRNA library (EPIKOL) targeting only genes encoding epigenetic regulators ( Extended Data Fig. 2d , e ) 35 . In this instance, we again found that sgRNAs targeting the EZH2 and EED genes (encoding two components of the PRC2 complex) as well as the ASH2L gene (encoding a COMPASS component) were enriched in the emerging mesenchymal populations ( Fig.…”

Epigenetic-focused CRISPR/Cas9 screen identifies ASH2L as a regulator of glioblastoma cell survival

“…Following antibiotic selection, transduced cells were cultured for 30 days by maintaining the sgRNA coverage at each population doubling. Using standard library preparation protocols, deep sequencing and analysis 33 , we compared the sgRNA composition in initial and final timepoints. Histogram of median normalized read counts for all sgRNA plotted for U373 and T98G cells confirmed normal distribution of log2-transformed normalized counts, indicating that no bias was introduced during cloning or transduction steps ( Figure 1F ).…”

Orthogonal targeting of KDM6A/B and HDACs mediates potent therapeutic effects in IDH1-mutant glioma

“…Retroviral particles from pMIG Bcl-xL (Addgene #8790), and lentiviral particles from pLenti6.3/TO/V5 containing IDH1 R132H , 13 or pLentiCRISPRv2 (Addgene #52961) were produced in 293T cells as previously described 18 . Further details are described in Supplementary Information .…”

“…were produced in 293T cells as previously described 18 . Further details are described in Supplementary Information.…”