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 interrogate the roles of epigenetic modifiers in cancer cells, we generated an epigenome-wide CRISPR-Cas9 knockout 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 and depletion of essential genes. We discovered novel epigenetic modifiers that regulate triple-negative breast cancer (TNBC) and prostate cancer cell fitness. We confirmed the growth-regulatory functions of individual candidates, including SS18L2 and members of the NSL complex (KANSL2, KANSL3, KAT8) in TNBC cells. Overall, we show that EPIKOL, a focused sgRNA library targeting ~800 genes, can reveal epigenetic modifiers that are essential for cancer cell fitness under in vitro and in vivo conditions and enable the identification of novel anti-cancer targets. Due to its comprehensive epigenome-wide targets and relatively high number of sgRNAs per gene, EPIKOL will facilitate studies examining functional roles of epigenetic modifiers in a wide range of contexts, such as screens in primary cells, patient-derived xenografts as well as in vivo models.

Chromatin-focused genetic and chemical screens identify BRPF1 as a targetable vulnerability in Taxol-resistant triple-negative breast cancer

“…RNA isolation and cDNA synthesis were performed as described [30]. List of qPCR primers can be found in Supplementary Table 2 .…”

“…Genomic DNA was isolated using Nucleospin Tissue kit (Macherey-Nagel, Germany) according to manufacturer’s instructions. Next generation sequencing (NGS) libraries were prepared as described [30]. Briefly, in the first PCR, 13.2 µg of gDNA was used as a template to achieve 250x coverage of the sgRNA library.…”

“…Genomic DNA was isolated using Nucleospin Tissue kit (Macherey-Nagel, Germany) according to manufacturer's instructions. Next generation sequencing (NGS) libraries were prepared as described [30].…”

“…Dual-color competition assays. For validation of EPIKOL screen candidate hits, dual color competition assays were performed as described [30]. Briefly, T1-160-Cas9 cells expressing either PGK-H2BmCherry (Addgene #21217) or PGK-H2BeGFP (Addgene #21210) were seeded as 50.000 cells/well in 12-well plates.…”

Ten Years of CRISPRing Cancers In Vitro

“…CRISPR technology has facilitated the generation of CRISPR/Cas9 knockout libraries, used to target epigenetic modifiers and to discover new chromatin-modifying genes responsible for different tumor fitness, like triple-negative breast and prostate cancer [ 82 ].…”