**3. CRISPR-Cas9 technology for disrupting fusion oncogenes**

Fusion oncoproteins arising from chromosomal rearrangements are known to drive the pathogenesis of a variety of hematological neoplasms such as CML, which results from a reciprocal translocation between chromosome 9 and 22 [30, 31]. This translocation fuses the *ABL1* gene on chromosome 9 to the *BCR* gene on chromosome 22, resulting in a *BCR/ABL* fusion gene, whose product is a cytoplasmic 210-KDa protein with upregulated tyrosine kinase activity that is considered essential for growth and survival of the leukemic cells [32]. As we previously mentioned, the discovery of *BCR-ABL*-mediated pathogenesis of CML provided the insight for the design of an inhibitory agent that targets BCR/ABL kinase activity such as imatinib mesylate. However, a substantial proportion of CML patients may not achieve the desired response or may eventually fail to respond adequately to these drugs [4]. A recent study of the BCR/ABL oncogen showed this gene fusion to be an ideal target for CRISPR/- Cas9-mediated gene therapy. A CRISPR-Cas9 application truncated the specific BCR-ABL fusion (p210) in an *in vitro* cellular model [15] (**Figure 6**).-

In this study, a nontumorigenic cell line (BaF3), which needs IL-3 to survive and proliferate- [33], was transformed with the fusion oncogene BCR/ABLp210 (BaF3-p210). The human- BCR/ABL oncogenic fusion confers on BaF3 the ability to survive and proliferate in the- absence of IL-3 and forms tumors in a xenograft model. Three custom-designed sgRNAs- were used to genetically inactivate the BCR/ABL oncogene. These specific sgRNAs directed- Cas9 to the BCR/ABL fusion sequence (Bcr-Abl sgRNA) or to the Abelson tyrosine kinase- sequence (Tk-Abl 1 sgRNA and Tk-Abl 2 sgRNA) (**Figure 7**). Lentiviral infection assays were- performed with each CRISPR/Cas9 reagents to generate three different BaF3-p210 cell lines- with the potentially edited BCR/ABL oncogene at the expected cleavage point in each one.-

**Figure 6.** Experimental model to show the ability of CRISPR/Cas9 to truncate BCR/ABL fusion. The non-tumorigenic and IL-3-dependent BaF3 cell line was transformed with fusion oncogene BCR/ABLp210. The transformed cell line is able to grow and survive in the absence of IL-3, although the cells enter into apoptosis when CRISPR/Cas9 introduces mutations in the sequence of the BCR/ABL oncogene, preventing its expression [15].

**Figure 7.** Schematic representation of BCR/ABL fusion transgene. Sequences of sgRNAs designed to edit fusion region (red boxes). One of them hybridizes at the BCR/ABL junction, and the other two hybridize in exon 2 of ABL [15].

The CRISPR/Cas9 system efficiently induced various mutations at the expected cleavage point, giving rise to three distinct BaF3-p210 cell lines (CRISPR-BaF3-p210) with several altered BCR/ABL sequences.-

As a result, significantly more cell death was observed in all CRISPR-BaF3-p210 cell lines in the absence of IL-3 than in BaF3 parental cells or mock BaF3-p210 cells (**Figure 8**).-

Xenograft experiments were carried out to determine whether the tumorigenic capacity was also blocked by the action of the CRISPR/Cas9 system. Mice injected with the three

**Figure 8.** Functional analysis of CRISPR-BaF3-p210-edited cells. Annexin V labeling was measured by flow cytometry in edited cells (BCR-ABL, TK-ABL1 and TK-ABL-2) in the presence and absence of IL-3. When IL-3 was removed from the medium, the three cell lines showed an increase in apoptosis, reflecting the absence of expression of the BCR-ABL oncogene [15].

CRISPR-BaF3-p210 cell lines gave rise to significantly smaller subcutaneous tumors than those produced by the nonedited cells (**Figure 9**).-

As expected, these small tumors were composed of nonedited cells, edited cells with +3/−3bp indels (or multiples), or cells with nonframeshift mutations. This result indicated that a specific cellular selection or more specific sgRNAs should be necessary before potential gene

**Figure 9.** *In vivo* effects of CRISPR-mediated editing of BCR/ABL oncogene. Tumor growth over 24days following subcutaneous cell injection. The final tumor mass was reduced by half in the CRISPR-BaF3-p210, relative to controls. CRISPR-BaF3 single cell-derived cell line (SC) cells were unable to form a subcutaneous tumor [15].

therapy in human. For this purpose, a CRISPR-BaF3-p210 cell line derived from a single cell (CRISPR-BaF3-p210-SC) carrying an 8-bp deletion (**Figure 9**) was selected to test tumorigenic capacity. No tumor growth was observed in any mouse injected with cells derived from the single-edited cell line (**Figure 9**).-
