Generation of conditional oncogenic chromosomal translocations using CRISPR–Cas9 genomic editing and homology‐directed repair

Chromosomal rearrangements encoding oncogenic fusion proteins are found in a wide variety of malignancies. The use of programmable nucleases to generate specific double‐strand breaks in endogenous loci, followed by non‐homologous end joining DNA repair, has allowed several of these translocations to...

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Bibliographic Details
Published in:The Journal of pathology 2017-05, Vol.242 (1), p.102-112
Main Authors: Spraggon, Lee, Martelotto, Luciano G, Hmeljak, Julija, Hitchman, Tyler D, Wang, Jiang, Wang, Lu, Slotkin, Emily K, Fan, Pang‐Dian, Reis‐Filho, Jorge S, Ladanyi, Marc
Format: Article
Language:English
Subjects:
Artificial Gene Fusion - methods
chromosomal translocations
Chromosomes, Human, Pair 11 - genetics
Chromosomes, Human, Pair 22 - genetics
Clustered Regularly Interspaced Short Palindromic Repeats - genetics
CRISPR–Cas9
Desmoplastic Small Round Cell Tumor - genetics
desmoplastic small round cell tumour (DSRCT)
DNA, Neoplasm - genetics
Ewing sarcoma
Gene Expression Regulation, Neoplastic - genetics
genomic editing
homology‐directed repair
Humans
oncogene
Oncogene Proteins, Fusion - genetics
Recombinational DNA Repair - genetics
Sarcoma, Ewing - genetics
Translocation, Genetic
Tumor Cells, Cultured
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Summary:Chromosomal rearrangements encoding oncogenic fusion proteins are found in a wide variety of malignancies. The use of programmable nucleases to generate specific double‐strand breaks in endogenous loci, followed by non‐homologous end joining DNA repair, has allowed several of these translocations to be generated as constitutively expressed fusion genes within a cell population. Here, we describe a novel approach that combines CRISPR–Cas9 technology with homology‐directed repair to engineer, capture, and modulate the expression of chromosomal translocation products in a human cell line. We have applied this approach to the genetic modelling of t(11;22)(q24;q12) and t(11;22)(p13;q12), translocation products of the EWSR1 gene and its 3' fusion partners FLI1 and WT1, present in Ewing's sarcoma and desmoplastic small round cell tumour, respectively. Our innovative approach allows for temporal control of the expression of engineered endogenous chromosomal rearrangements, and provides a means to generate models to study tumours driven by fusion genes. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
ISSN:0022-3417
1096-9896
DOI:10.1002/path.4883