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

Abstract

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.

Keywords: CRISPR-Cas9; Ewing sarcoma; chromosomal translocations; desmoplastic small round cell tumour (DSRCT); genomic editing; homology-directed repair; oncogene.

Figure 1. Generation of a de novo EWSR1-FLI1 t(22;11)(q12:q24) chromosomal translocation via CRISPR-Cas9 and NHEJ

A. Schematic of the t(22;11)(q12:q24) rearrangement mediated by CRISPR-Cas9 genomic editing and non-homologous end-joining (NHEJ). gRNAs were designed to target intron 7 (E4) and intron 5 (F3) of EWSR1 and FLI1 loci, respectively. B. RT-PCR confirming specific expression of the EWSR1-FLI1 fusion transcript between exon 7 of EWSR1 and exon 6 of FLI1 (top panel). TC71: fusion positive Ewing's sarcoma patient derived cell line. NT: non-transfected control. Sanger sequencing trace encompassing the fusion event is shown (lower panel). C. Confirmation of the translocation at the genomic level. NT: non-transfected control. Sanger sequencing traces encompassing the genomic fusion event are shown (lower panel). Asterisk denotes the insertion of one additional nucleotide by NHEJ.

Figure 2. Generation of the EWSR1-FLI1 t(11;22)(q24;12) chromosomal translocation via CRISPR-Cas9 using homology-directed repair (HDR)

A. Schematic of the generation of the t(11;22)(q24:q12) translocation using CRISPR-Cas9 genomic editing and homology-directed repair (HDR). The introduction of a NG1-EWSR1-FLI1 (NG1-EF) targeting vector, which bridges the Cas9-gRNA directed double-strand breakpoints in EWSR1 and FLI1 loci, leads to the formation of a chimeric translocation, NG1-EWSR1-FLI1 t(11;22)(q24;12). The NG1-EF targeting vector contains arms of homology matching to EWSR1 and FLI1 (shaded, LHA and RHA). B. Schematic representation of the 2-step PCR screening strategy. The external EWSR1 and FLI1 primers are specific for genomic DNA outside of the sequence retained in the left and right homology arms of the NG1-EF targeting vector (LHA and RHA, shaded areas). Treatment with Cre recombinase removes the selectable cassette leaving a single LoxP at the engineered locus. C. A 2-Step PCR screening protocol on genomic DNA isolated from puromycin resistant clones identifies correctly targeted NG1-EF clones. Positioning of the primers is depicted in B. Combinational PCR screening using primers: (1) EWSR1-FW with Puro-RV (2) TK-FW with FLI1-RV identifies correctly targeted clones that contain both the left and the right homology arm and hence the NG1-EWSR1-FLI1 hybrid translocation. D. Following Cre recombination and ganciclovir selection, genomic DNA was assayed to demonstrate the loss of the selectable cassette and the generation of the EWSR1-LoxP-FLI1 translocation by using a EWSR1-FW primer and a FLI1-RV primer.

Figure 3. Faithful expression of a biologically active EWSR1-FLI1 translocation product engineered with CRISPR-Cas9 and HDR

A. Verification of the EWSR1-FLI1 fusion by FISH. The EWSR1 locus-specific probe labelled in red, and the FLI1 locus-specific probe labelled in green. In the parental cells, distinct separate green and red signals were observed. In cells from the NG1-EF-16.2 and NG1-EF-57.4 clones, green/red fusion signals (appearing as partial or complete yellow dots) that represented EWSR1-FLI1 fusions were identified. DNA was counterstained with DAPI (Blue). B. RT-PCR confirming specific expression of the EWSR1-FLI1 fusion transcript in the NG1-EF16.2 and NG1-EF57.4 engineered clones, but not in the parental cell line. Sanger sequencing trace encompassing the fusion event is shown (lower panel) confirming the EWSR1-FLI1 fusion mRNA. TC71: Fusion positive patient-derived Ewing's sarcoma cell line. C. Expression of EWSR1-FLI1 fusion protein in the NG1-EF16.2 and NG1-EF57.4 clones as detected by an anti-FLI1 C-terminal antibody (top panel). The TC71 Ewing's sarcoma cell line was used a positive control. GAPDH was used as loading control (bottom panel). * non-specific band D. Expression of EWSR1-FLI1 regulated transcripts in NG1-EF16.2 and NG1-EF57.4 clones as established by RT-qPCR. Significant upregulation of expression of NROB1, Cyclin D1 and NKX2.2 in NG1-EF16.2 and NG1-EF57.4 clones compared to the parental cell line. Data are presented as relative expression of a target transcript compared to the parental cell line. The data are derived from a total of 3 independent experiments (mean ± SD).

Figure 4. Generation of conditional endogenous EWSR1-FLI1 t(11;22)(q24;q12) chromosomal translocation via CRISPR-Cas9, HDR and inclusion of a gene-trap

A. Schematic representation of a conditional chromosomal translocation with the incorporation of a gene-trap (TagRFP-Trap) in the engineered hybrid intron. B. Isolation of TagRFP positive clones by FACS following HDR targeting with the NG2-EWSR1-FLI1 HDR template and co-transfection of EWSR1 and FLI1 gRNA and 6 days of puromycin selection. C. Schematic representation of the primer positions used in the 2-step PCR screen (Top panel). The external EWSR1 and FLI1 primers are specific for genomic DNA outside of the sequence retained in the left and right homology arms of the NG2-EF-TagRFP targeting vector (shaded areas), whilst the internal primers were specific for TagRFP (RFP) and thymidine kinase (TK). Combinational PCR screening using primers in: (1) EWSR1-FW with a TagRFP-RV specific primer and (2) TK-FW primer with a FLI1-RV primer identifies correctly target clones that contain both the left and the right homology arm and hence the NG2-EWSR1-FLI1 hybrid translocation. D. RT-PCR analysis confirms that the NG2-EF-TagRFP clones express the EWSR1-TagRFP transcript at the expense of the full-length EWSR1-FLI1 mRNA, E. RT-PCR analysis confirms expression of the EWSR1-FLI1 fusion transcript in NG2-EF-TagRFP clones only in the presence of transiently expressed Cre recombinase, which excises the TagRFP-Trap, allowing for the transcription of the full-length fusion EWSR1-FLI1 mRNA.

Figure 5. Generation of an conditional de novo EWSR1-WT1 t(11;22)(p13;q12) translocation, the hallmark of desmoplastic small round cell tumour (DSRCT)

A. Schematic representation of the generation of a conditional EWSR1-WT1 chromosomal translocation using the incorporation of a gene-trap (TagRFP-Trap) at the engineered intronic junction between the CRISPR-Cas9-mediated translocation of EWSR1 to WT1. gRNAs were directed against Intron 7 of EWSR1 and Intron 7 of WT1. B. Schematic representation of the primer positions used in the 2-step PCR screen (top panel). The external EWSR1-FW and WT1-RV primers are specific for genomic DNA outside of the sequence retained in the left and right homology arms of the NG2-EWSR1-WT1 targeting vector (shaded areas), whilst the internal primers were TagRFP and thymidine kinase (TK). Combinational PCR screening using primers in: (1) EWSR1-FW with a TagRFP-RV specific primer and (2) TK-FW with a WT1-RV primer combination identifies a correctly targeted clone that contains both the left and the right homology arm and hence the EWSR1-NG2-WT1 hybrid translocation (lower panel); C. RT-PCR analysis confirms that the NG2-EW-TagRFP-4 clone expresses the EWSR1-TagRFP transcript at the expense of the full-length EWSR1-WT1 mRNA. D. RT-PCR analysis confirms expression of the EWSR1-WT1 fusion transcript in NG2-EW-TagRFP-4 clone only in the presence of transiently expressed Cre recombinase, which excises the TagRFP-Trap, releasing transcription of the full-length EWSR1-WT1 fusion mRNA. Sanger sequencing (lower panel) confirms authenticity of the EWSR1-WT1 fusion mRNA. E. Western Blot analysis confirms expression of EWSR1-WT1 protein following transient expression of Cre recombinase. F. RT-qPCR confirms up-regulation in expression of ASCL1 following Cre-mediated activation of EWSR1-WT1. Data are presented as relative expression of a target transcript compared to the parental cell line. The data are derived from a total of 3 independent experiments (mean ± SD).