The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia

Abstract

CRISPR/Cas9 technology was used to abrogate p210 oncoprotein expression in the Boff-p210 cell line, a pro-B line derived from interlukin-3-dependent Baf/3, that shows IL-3-independence arising from the constitutive expression of BCR-ABL p210. Using this approach, pools of Boff-p210-edited cells and single edited cell-derived clones were obtained and functionally studied in vitro. The loss of p210 expression in Boff-p210 cells resulted in the loss of ability to grow in the absence of IL-3, as the Baf/3 parental line, showing significantly increased apoptosis levels. Notably, in a single edited cell-derived clone carrying a frame-shift mutation that prevents p210 oncoprotein expression, the effects were even more drastic, resulting in cell death. These edited cells were injected subcutaneously in immunosuppressed mice and tumor growth was followed for three weeks. BCR/ABL-edited cells developed smaller tumors than those originating from unedited Boff-p210 parental cells. Interestingly, the single edited cell-derived clone was unable to develop tumors, similar to what is observed with the parental Baf/3 cell line.CRISPR/Cas9 genomic editing technology allows the ablation of the BCR/ABL fusion gene, causing an absence of oncoprotein expression, and blocking its tumorigenic effects in vitro and in the in vivo xenograft model of CML. The future application of this approach in in vivo models of CML will allow us to more accurately assess the value of CRISPR/Cas9 technology as a new therapeutic tool that overcomes resistance to the usual treatments for CML patients.

Etv6/Runx1 Fusion Gene Abrogation Decreases The Oncogenic Potencial Of Tumour Cells In A Preclinical Model Of Acute Lymphoblastic Leukaemia

“…One of them directed towards the end of exon 5 of ETV6 and other directed towards the beginning of intron 5-6, both before the fusion point, with the intention of producing indels or deletions that modify the open reading frame of the oncogene, and, therefore, the gene expression. These sgRNAs were cloned into a vector containing the Cas9 nuclease coding sequence and GFP, pSpCas9(BB)-2A-GFP (PX458) (Addgene plasmid #48138) 15 as described previously 14 (Supplementary Table 1). Then they were electroporated into the REH cells.…”

“…The implementation of new genetic editing strategies has allowed the development of functional studies by generation of gene and gene fusion KO models, both in vitro and in vivo. 14 In this study, we completely abrogated the expression of E/R fusion protein in REH ALL cell line using the CRISPR/Cas9 editing system and we studied the effects of genetic ablation of the fusion protein in cellular functions. We also studied whether the suppression of E/R expression sensitize tumour cells to PI3K inhibitors.…”

Application of CRISPR/Cas9 gene editing technique in the study of cancer treatment

“…in vitro studies have also shown that CRISPR/Cas9 can effectively inhibit tumor cell growth by targeted deletion of oncogenes. [32][33][34] Oncogenes are important targets in CRISPR/Cas9 applied for the treatment of tumors. They can be targeted by CRISPR/Cas9 to knockout oncogenes, interfere with the expression of related proteins, affect the activity of oncogenes, and further inhibit tumor growth.…”

Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency

“…Unlike ribozymes, antisense oligodeoxynucleotides (AS-ODNs) and short interfering RNAs (siRNAs), CRISPR/Cas9 works at the DNA level, where it has the advantage of providing permanent and full gene knockout, while other methods only silence genes transiently(2) , (3). CRISPR/Cas9 cuts DNA in a sequence-specific manner with the possibility of interrupting coding sequences, thereby making it possible to turn off cancer drivers in a way that was not previously feasible in humans (4,5). This notable application of permanent gene disruption is based on the cellular mechanisms involved in double-stranded break (DSB) repair.…”

“…A recent study of the BCR/ABL oncogene 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) abrogating its oncogenic potential, but to achieve in vivo effectiveness in a xenograft model, the authors had to select and expand the correctly edited cellular clone because some of the clones contained in-frame or nonsynonymous mutations (5,15). Therefore, in these situations, it is essential to have not only highly efficient Cas9-sgRNA cell delivery, but also a high capacity for generating null mutations.…”