Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing

Abstract

Genome sequencing studies have shown that human malignancies often bear mutations in four or more driver genes, but it is difficult to recapitulate this degree of genetic complexity in mouse models using conventional breeding. Here we use the CRISPR-Cas9 system of genome editing to overcome this limitation. By delivering combinations of small guide RNAs (sgRNAs) and Cas9 with a lentiviral vector, we modified up to five genes in a single mouse hematopoietic stem cell (HSC), leading to clonal outgrowth and myeloid malignancy. We thereby generated models of acute myeloid leukemia (AML) with cooperating mutations in genes encoding epigenetic modifiers, transcription factors and mediators of cytokine signaling, recapitulating the combinations of mutations observed in patients. Our results suggest that lentivirus-delivered sgRNA:Cas9 genome editing should be useful to engineer a broad array of in vivo cancer models that better reflect the complexity of human disease.

FIGURE 1. Stable modification of hematopoietic stem cells by a lentiviral sgRNA:Cas9 delivery system

A) Depiction of a lentiviral vector for bi-cistronic expression of the sgRNA from a U6 promoter (U6) and cas9 from a short EF1a promoter (EFS) with a fluorescent protein marker (eGFP) from a picorna virus derived 2A auto-cleavage site (P2A). A lentiviral vector that allows incorporation of target sites into the coding sequence of a fluorescent protein (RFP657) is depicted at the bottom. A Puromycin resistance gene (PAC) is expressed from an internal ribosomal entry site (IRES) B) Super-transduction of a reporter cell line with an eGFP tagged sgRNA:Cas9 vector targeting the respective site (blue) induces loss of reporter fluorescence. A non-targeting sgRNA:Cas9 vector (red) does not affect reporter fluorescence. Quantification by plotting the MFI of the reporter in targeting (blue) vs non-targeting (red) sgRNA transduced cells (black box, left plot). C) Efficacy of spacers for recurrently mutated genes in myeloid malignancy. Efficacy was assessed with the RFP657 reporter system described in A) and B). Results were normalized to non-targeting spacer. Spacer marked in red were used in following experiments. D) Surveyor assay based confirmation of target site cleavage at genomic loci for Tet2 and Runx1 spacers indicated in panel C compared to non-targeting spacer. Percentages above are quantified cleavage efficacies. E) Peripheral blood sgRNA:Cas9 vector marking tracked over a period of 19 weeks. A significant increase of sgRNA:Cas9 expressing cells with a spacer targeting Runx1 (n=4) was observed in comparison to mice expressing a non-targeting spacer (n=4). F) Stable expression of the sgRNA:Cas9 vector after 19 weeks in hematopoietic stem and progenitor cells. Overlay of eGFP expression in sgRNA:Cas9 transduced cells (blue) and non-transduced cells (red) of the respective cell population. MPP=Multipotent progenitors; ST-HSC=short-term HSCs; LT-HSC=long-term HSCs; LT-HSC CD34-=most quiescent long-term-HSCs

FIGURE 2. Multiplex gene targeting induces clonal development in vivo

A) Schematic for multiplex gene targeting with two lentiviral vectors: a sgRNA:Cas9-eGFP vector expressing sgRNA, Cas9 and eGFP, and a vector expressing additional sgRNAs with a RFP657 fluorescent reporter gene from a short EF1a promoter. B) Multiplexed gene targeting in mice (n=5) significantly increases myelomonocytic cells (CD11b+), decreases B-cells (B220+) and increases white blood cell counts at 12 weeks post-transplant in comparison to mice that were transplanted with control sgRNA vector transduced cells (n=4). C) Peripheral blood sgRNA:Cas-eGFP vector expression (x-axis) and expression of a pool of vectors expressing RFP657 (y-axis) over time starting 4 weeks post-transplant. Clonal populations were detected by the pattern of eGFP/RFP657 expression. Two clones were isolated (red gates indicate clones) and genomic regions of targeted genes were sequenced (displayed in red box). Mono- and bi-allelic deletions in Tet2, Dnmt3a, Nf1 and Runx1 were detected in one case. Detected mutations at 8 weeks are shown below. Sequences of the deletions are shown in Supplementary Figure 8A. D) Another clone was analyzed as in panel C and was found to have mutations in Tet2, Ezh2, Nf1 and Runx1 B-II. Detected mutations at 8 weeks are shown below. Sequences of the deletions are shown in Supplementary Figure 8B.

FIGURE 3. Myeloid malignancy modeling with multiplex genome editing

A) Flow cytometric analysis of a diseased C57Bl/6 mouse for expression of the introduced Cas9:sgRNA vectors and for myeloid (Gr1+/CD11b+). Detected mutations are shown in the right panel (displayed in red box). Sequence alignments are shown in Supplementary Figure 9. B) Peripheral blood smear (left; May-Gruenwald/Giemsa (MGG); 1000x) and BM (right; Hematoxylin-Eosin(HE), 1000x) showing granulocytic cells and blasts. Scale bar (5µm) given in the lower left. C) Western blots of Ezh2, Smc3 and activated Erk1/2 (pErk) protein levels in the leukemic BM compared to normal C57Bl/6 wild-type BM. D) Representative flow cytometry of secondary transplant recipient mouse peripheral blood. E) Lineage distribution in peripheral blood from secondary mice 8 weeks post-transplant (n=5). F) Histopathology analysis of BM (MGG; 1000×) from a secondary mouse that succumbed to leukemia. Scale bar (5µm) given in the lower left. G) Heatmap summarizing mutations detected in all mice. Each column presents clone. Targeted genes are presented in rows. Color legend is given below. A table indicating where individual clones have been presented is given on the right (Supplementary Figure abbreviated as S. Figure).