Editors' ChoiceCancer

A CRISPR Way to Move Things Around

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Science Translational Medicine  02 Jul 2014:
Vol. 6, Issue 243, pp. 243ec113
DOI: 10.1126/scitranslmed.3009626

When chromosomal parts are exchanged between nonhomologous chromosomes, a translocation is born. This is a very common event involved in the development of several cancers, especially hematological malignancies and sarcomas. The exact mechanism is unknown, but the starting event is thought to be DNA damage, which causes double-strand breaks, followed by “illegitimate joining” of elements from two different chromosomes. Engineering these translocations into cell models has been limited by the availability of flexible and user-friendly genome editing tools.

The recent expansion of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems, which can cut the genome at any desired location, gave Torres et al. an opportunity to recreate two translocations with this method. The first was the t(11;22)(q24;q12) chromosomal translocation, which generates the EWSR1-FLI1 fusion gene, a driving event for Ewing’s sarcoma. The second translocation they generated was t(8;21)(q22;q22), which joins the RUNX1 gene on chromosome 21 with the ETO gene on chromosome 8 in acute myeloid leukemia.

The authors started by targeting the gene partners in the t(11;22) translocation with small guiding RNAs and confirmed that the RNA-guided endonuclease induced double-strand breaks at the sites of selected genes on two different chromosomes. The investigators transfected human embryonic kidney (HEK) 293A cells with different small guiding RNA combinations and were able to show that a translocation occurred using fluorescence in situ hybridization (FISH) with an EWSR1 single-fusion probe. A polymerase chain reaction also confirmed the production of a fusion gene. The resulting fusion protein was functional and affected the transcription of known target genes. The team then successfully used the same CRISPR/Cas9 strategy to generate the t(11;22) translocation in genetically unmodified human mesenchymal stem cells, showing the effectiveness of this technique in noncancerous human cells, which will allow future studies of the early effects of these translocations. The same technique was used to induce t(8;21) translocations, first in HEK293A cells and then in human CD34+ human hematopoietic/progenitor stem cells.

With this method of recreating translocations common in human cancers, researchers now have a new and “user-friendly” tool to study both the mechanisms and the effects of these translocations in oncogenesis.

R. Torres et al., Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR–Cas9 system. Nat. Commun. 5, 3964 (2014). [Abstract]

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