In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system

Abstract

Chromosomal rearrangements play a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions1. A recently discovered example is a fusion between the Echinoderm Microtubule-associated Protein-like 4 (EML4) and the Anaplastic Lymphoma Kinase (ALK) genes, generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4-ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC)2 and is clinically relevant because it confers sensitivity to ALK inhibitors3. Despite their importance, modeling such genetic events in mice has proven challenging and requires complex manipulation of the germline. Here we describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. We apply it to generate a mouse model of Eml4-Alk-driven lung cancer. The resulting tumors invariably harbor the Eml4-Alkinversion, express the Eml4-Alk fusion gene, display histo-pathologic and molecular features typical of ALK+ human NSCLCs, and respond to treatment with ALK-inhibitors. The general strategy described here substantially expands our ability to model human cancers in mice and potentially in other organisms.

CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice

“…One potential limitation of multiplexed CRISPR/Cas9 mutagenesis is that, in principle, combinatorial sgRNA targeting could lead to undesired large chromosomal rearrangements (18,20). To examine this possibility, we performed PCR-based screening for all possible deletions at chromosomes that were targeted by multiple sgRNAs (SI Appendix, Fig.…”

“…This break is repaired by nonhomologous end joining, which commonly leaves a short insertion or deletion (indel), allowing homozygous disruption of the targeted gene. Recent studies showed that CRISPR/Cas9 is functional in germ cells and somatic cells of mice and can be used for gene editing and cancer induction in the lung and the biliary compartment (14)(15)(16)(17)(18)(19)(20). Targeting of Pten and p53 in the liver was reported to induce Significance Assigning biological relevance and molecular function to large catalogues of mutated genes in cancer is a major challenge.…”

CRISPR, the disruptor

“…Her worries began at a meeting in 2014, when she saw a postdoc present work in which a virus was engineered to carry the CRISPR components into mice. The mice breathed in the virus, allowing the CRISPR system to engineer mutations and create a model for human lung cancer 4 . Doudna got a chill; a minor mistake in the design of the guide RNA could result in a CRISPR that worked in human lungs as well.…”

Excitation et crispations autour de CRISPR : lorsque la réalité dépasse la science-fiction

“…Beaucoup moins vont audelà pour analyser les conséquences biologiques des modifications introduites. Parmi les réalisations les plus significatives dans le domaine biomédical voici quelques exemples spectaculaires donnés dans l'ordre chronologique : -2014 : obtention de réarrangements chromosomiques reproduisant des situations observées en oncologie 23 ; -2015 : élaboration de la méthode MCR (Mutagenic Chain Reaction) où une modification hétéro-zygote agit en trans de manière autocatalytique pour obtenir l'homozygotie en court-circuitant la transmission mendélienne 24 . Ce procédé d'abord validé chez la Drosophile a été appliqué chez l'Anophèle vecteur de l'agent du paludisme.…”