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Harvard Team Publishes Details of CRISPR, RNAi Combo Screening Approach

NEW YORK (GenomeWeb) – A team from Harvard Medical School this week published the details of a novel approach to drug-target screening, combining the genome-editing technology CRISPR and the gene-silencing technology RNAi to identify three genes that appear to be essential to the growth of tumor cells but not normal cells.

According to the researchers, the method offers a number of advantages over conventional RNAi screening, which is limited by incomplete transfection, partial knockdown, and off-target effects, and therefore often requires secondary screening.

In their study, which appeared in Science Signaling, the scientists focused on the tuberous sclerosis complex (TSC), a disorder characterized by the growth of benign tumors on the brain and other vital organs. It is caused by mutations in one of two tumor suppressors, TSC1 and TSC2, and is currently treated with chemotherapeutic agents.

RNAi screening for new drug targets routinely involves the use of multiple RNAi reagents against individual genes in order to ensure robust target knockdown. This, however, can result in increased off-target effects and variable knockdown efficiencies, which in turn leads to high false-positive and false-negative rates, the Harvard team noted.

As a result, extensive secondary screening and validation is often required, making the overall approach expensive and time-consuming.

To overcome this issue, the researchers used CRISPR to create isogenic mutant Drosophila cells lines deficient in either TSC1 or TSC2, which were then used in single-reagent RNAi screens against all of the insect's kinases and phosphatases. The knockdown of three genes in particular showed robust and specific effects on TSC1- and TSC2-deficint cells, including a reduced growth rate, but not in wild-type cells.

When the genes were knocked down in murine and human tumor-derived TSC2-deficient cell lines, a similar growth-inhibiting effect was seen, "illustrating the power of this cross-species screening strategy to identify potential drug targets," the study's authors wrote.

Overall, the use of CRISPR-generated mutant cell lines with single RNAi reagents sidestepped much of the noise associated with dual RNAi-based screening approaches, the researchers noted. Further, the use of homogeneous populations of null mutant cells avoids the issues of incomplete transfection and incomplete knockdown.

The investigators also highlighted the potential for mutant cell lines as higher-quality disease models compared with RNAi-mediated knockdowns. "For example, diseases such as TSC are caused by loss-of-function mutations rather than by partial transient reduction in protein abundance," they wrote. "The establishment of a mutant cell line enables cellular adaptation to the induced mutation, likely generating a more representative cellular environment."

Given the robustness of the method, as evidenced by the conservation of the identified synthetic interactions between mouse and human systems, the study's authors expect it to be generally applicable to various other biological and disease-relevant questions.