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CRISPR/Cas9 Used to Model Translocations

NEW YORK (GenomeWeb) – Horizon Genomics, a subsidiary of Horizon Discovery Group, has published a study describing its ability to generate fusion oncogenes in cell lines or undo them and revert the cell to its wild-type state.

Led by senior author Tilmann Bürckstümmer, the scientists at the cell line engineering firm showed how they were able to generate de novo CD74-ROS1 fusions and undo BCR-ABL1 fusions in the eHAP human haploid cell line using CRISPR/Cas9.

To create and undo the fusions, the scientists designed two guide RNAs to create DSBs at the required locations, and screened hundreds of clones using PCR primers covering the targeted genomic junction. They said they generated CD74-ROS1 translocations at just over 1 percent efficiency, while they were able to undo BCR-ABL1 fusions just under 1 percent of the time.

They published their results Sept. 16 in BMC Genomics.

Genetic abnormalities are a hallmark of cancer cells and translocations can be driver mutations in a number of cancer types, "yet definitive experimental models are missing," the authors wrote. "The nature of gene fusions strongly correlates with the tumor type, making them very attractive targets for cancer diagnostic or therapeutic intervention."

Because CRISPR/Cas9 can easily make targeted double-strand breaks, several teams of scientists have already been able to create cell lines with specific translocations. However, the Horizon team wrote that "the overall efficiency by which clones were retrieved was not always apparent and the resulting cell lines were poorly characterized."

In addition to PCR-based analysis to detect and characterize the fusions, the researchers also used RT-PCR to verify they were detectable as mRNAs. Spectral karyotyping showed that the cells did not carry additional genomic abnormalities.

While the main experiments were done using a haploid cell line, the scientists said getting the same results in diploid cells should be "equally feasible." They also created CD74-ROS1 and its reciprocal translocations in HEK293T cells.

The authors further suggested that CRISPR-engineered cell lines could serve as a source of reference standard-grade genomic DNA or mRNA for cancer diagnostics. "Implementation of such reference standards is likely to increase the quality of diagnostic output and will thus represent an important advancement in the era of personalized medicine," they wrote.