Modeling of the Human Alveolar Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nuclease

Abstract

Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.

A Review of CRISPR-Based Genome Editing: Survival, Evolution and Challenges

“…The delivery approaches vary depending on application method and the cell type (Ahmad and Amiji, 2018). For instance, plasmids carrying Cas9 and sgRNA are conveyed through transitory transfection methods for small scale systematic application in cells, such as nucleofection, lipofectamine, and electroporation Lagutina et al, 2015), because incessant expression is not required once the preferred genome manipulation is achieved. However, transitory transfection might not be appropriate for repression analyses because CRISPRi only affects transcriptional efficiency relative to both protein and mRNA constancy (Carrol et al, 2016;Yang et al, 2016).…”

Modeling Disease In Vivo With CRISPR/Cas9

“…In some cases, reciprocal translocations observed in human disease are not easily created in the mouse due to differences in respective gene orientation and the consequent production of potentially unstable dicentric chromosomes. To circumvent this limitation, Lagutina et al , took a clever two-step approach, first inverting a large syntenic region on mouse chromosome 3 through a Cre-LoxP strategy, then using CRISPR to catalyse the translocation of two transcription factors, Pax3 and Foxo1, frequently observed in rhabdomyosarcoma [53]. These studies are impressive examples of how CRISPR/Cas9 tools can enable the interrogation of structural genomic variants and the rapid production of tailored animal models for pre-clinical testing.…”

CRISPR/Cas9-Facilitated Chromosome Engineering to Model Human Chromosomal Alterations

“…The most genetically accurate model among them is the line carrying triplications spanning the entirety of all Hsa21 syntenic regions, Dp (10)1Yey/+;Dp (16)1Yey/+;Dp (17)1Yey/+ [4]. Phenotypic characterizations of this mutant revealed several important DS-related phenotypes, including heart defects and impaired cognitive function.…”

“…Other engineered mouse mutants, which carry a triplication or deiciency of smaller Hsa21 syntenic regions [5,6], have facilitated systematic genetic dissections of DS phenotypes. With the emergence of CRISPR/Cas9-facilitated genome editing, atempts have been made to further improve the eiciency of mammalian chromosome manipulations, whether it be deletions, duplications, inversions, or translocations [7][8][9][10], including those in Hsa21 syntenic regions.…”