Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system

R Torres; M C Martin; A Garcia; Juan C Cigudosa; J C Ramirez; S Rodriguez-Perales

doi:10.1038/ncomms4964

Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system

Nat Commun. 2014 Jun 3:5:3964. doi: 10.1038/ncomms4964.

Authors

R Torres¹, M C Martin², A Garcia¹, Juan C Cigudosa², J C Ramirez¹, S Rodriguez-Perales²

Affiliations

¹ Viral Vector Facility, Fundacion Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernandez Almagro 3, 28029 Madrid, Spain.
² Molecular Cytogenetics Group, Spanish National Cancer Centre-CNIO, Melchor Fernandez Almagro 3, 28029 Madrid, Spain.

PMID: 24888982
DOI: 10.1038/ncomms4964

Abstract

Cancer-related human chromosomal translocations are generated through the illegitimate joining of two non-homologous chromosomes affected by double-strand breaks (DSB). Effective methodologies to reproduce precise reciprocal tumour-associated chromosomal translocations are required to gain insight into the initiation of leukaemia and sarcomas. Here we present a strategy for generating cancer-related human chromosomal translocations in vitro based on the ability of the RNA-guided CRISPR-Cas9 system to induce DSBs at defined positions. Using this approach we generate human cell lines and primary cells bearing chromosomal translocations resembling those described in acute myeloid leukaemia and Ewing's sarcoma at high frequencies. FISH and molecular analysis at the mRNA and protein levels of the fusion genes involved in these engineered cells reveal the reliability and accuracy of the CRISPR-Cas9 approach, providing a powerful tool for cancer studies.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Artificial Gene Fusion
CRISPR-Cas Systems*
Calmodulin-Binding Proteins / genetics
Core Binding Factor Alpha 2 Subunit / genetics
DNA Breaks, Double-Stranded*
Humans
In Vitro Techniques
Leukemia, Myeloid, Acute / genetics*
Oncogene Proteins, Fusion / genetics*
Oncogene Proteins, Fusion / metabolism
Proto-Oncogene Protein c-fli-1 / genetics
Proto-Oncogene Proteins / genetics
RNA, Guide, CRISPR-Cas Systems*
RNA, Messenger / metabolism*
RNA-Binding Protein EWS
RNA-Binding Proteins / genetics
RUNX1 Translocation Partner 1 Protein
Sarcoma, Ewing / genetics*
Transcription Factors / genetics
Translocation, Genetic / genetics*

Substances

Calmodulin-Binding Proteins
Core Binding Factor Alpha 2 Subunit
EWSR1 protein, human
FLI1 protein, human
Oncogene Proteins, Fusion
Proto-Oncogene Protein c-fli-1
Proto-Oncogene Proteins
RNA, Guide, CRISPR-Cas Systems
RNA, Messenger
RNA-Binding Protein EWS
RNA-Binding Proteins
RUNX1 Translocation Partner 1 Protein
RUNX1 protein, human
RUNX1T1 protein, human
Transcription Factors