CRISPR-SID: identifying EZH2 as a druggable target for desmoid tumors viain vivodependency mapping

Abstract

Identification of true dependencies in cancer is pivotal to the elucidation of novel therapeutic strategies to increase prospects for cancer patients. Unfortunately, in vivo identification of genetic dependencies has long relied on expensive and time-consuming breeding of genetically engineered animal models.Recently, in vitro CRISPR/Cas9 screens provided a new method for rapid and genome-wide identification of genetic dependencies. Nevertheless, genetic dependencies would ideally be identified using in vivo cancer models initiated by clinically relevant oncogenic driver or tumor suppressor insults. Here, we report a new methodology called CRISPR/Cas9-mediated Negative Selection Identification of genetic Dependencies (CRISPR-NSID) that allows in vivo elucidation of cancer cell vulnerabilities in genetic cancer models. The methodology hinges on the fact that for a genetic dependency there is an incapability for recovering tumors carrying biallelic frameshift mutations in this gene. We demonstrate how integrating experimentally determined, or in silico predicted, probabilities of frameshift editing for any given gRNA can be employed to ascertain negative selection pressure on inactivation of a genetic dependency during tumorigenesis. As a proof-of-principle, we use CRISPR-NSID to identify ezh2 and creb3l1 as genetic dependencies in desmoid tumors (desmoid-type fibromatosis) occurring in a Xenopus tropicalis cancer model driven by apc mutations. Bridging CRISPR-NSID to a clinically unmet need, we further demonstrate the promise for EZH2 inhibition as a new therapeutic strategy for desmoid tumors.This study establishes a new methodology for rapid identification of genetic dependencies in monoclonal disorders with wide adaptability to other model systems and organisms.

Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos

“…We had previously observed significant correlation between experimental ezh2 CRISPR/Cas9 gene editing outcomes in X. tropicalis embryos, and the predictions obtained for the InDelphi model trained in mouse embryonic stem cell modus (further abbreviated as: InDelphi-mESC) ( Fig. S1) 31 . We next questioned whether this observation would hold true in a larger dataset and also for other recently reported prediction models.…”

“…Implementation of InDelphi-mESC in the gRNA design process should facilitate the generation of frameshift heterozygous mutant F 1 animals upon out breeding, and potentially accelerate the creation of homozygous mutant F 1 animals by directly breeding F 0 founders. In addition, genome editing outcome predictions are useful to determine both positive and negative selection pressure on cellular fitness occurring as a consequence of underlying biological processes, such as-but not limited to-tumorigenesis and cancer 31,35 .…”

“…One-or two-days post injection genomic DNA was extracted from whole embryos (GenElute Mammalian Genomic DNA Miniprep Kit, Sigma-Aldrich, St. Louis, MO), the target regions were PCR amplified (Taq DNA Polymerase, New England Biolabs, Ipswich, MA) using primers indicated in supplementary table S1B, the PCR products were cleaned (EconoSpin Spin Column For DNA, Epoch Life Science, Missouri City, TX) and sent for Sanger sequencing (GENEWIZ, South Plainfield, NJ). For the zebrafish experiments, single-guide RNA (sgRNA) molecules were designed with the CRISPRdirect software (https :// crisp r.dbcls .jp/)36, and were produced as previously reported 31 . Briefly, in vitro transcription (MEGAshortscript T7 Transcription Kit, Invitrogen, cat.…”

“…AM1908) and quantity and integrity assessment. One-cell stage zebrafish embryos were injected with CRISPR/Cas9 components in the cell, as previously described 31 . At 1-day post fertilization (dpf), DNA extraction was performed on a pool of 20 embryos (KAPA Express Extract DNA Extraction Kit, Kapa Biosystems, KK7103).…”