New Multiplex Gene Interaction Mapping Technique Unveiled

New Multiplex Gene Interaction Mapping Technique Unveiled
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A new multiplex technique based on CRISPR-Cas has been developed at Goethe University Frankfurt that allows genetic interaction mapping with a fraction of the effort and cost of similar experiments, the researchers report. The technique makes it possible to introduce, remove and switch off genes in a targeted way using single guide RNA.

Their work appears in Nucleic Acids Research.

Combinatorial CRISPR-Cas screens have advanced genetic interaction mapping, but the cost and effort of such experiments is very high. As the Goethe researchers write: “the required experimental scale for GI screens can limit the number of simultaneously investigated gene combinations. Three parameters determine the scale, robustness and reproducibility of CRISPR screens: library diversity, library distribution skew, and experimental coverage.”

They say their new technique reduces the work involved, and the cost, by a factor of ten. The team reports they can produce the address libraries very uniformly and in high quality thanks to the new 3Cs multiplex technique. “Due to the mediocre quality of the CRISPR-Cas libraries previously available, very large experiments always had to be carried out to statistically compensate for any errors that arose,” says Manuel Kaulich, senior author of the report.

The scientists, from the Institute of Biochemistry II at Goethe University, expanded the 3Cs technique that they developed and patented three years ago. 3Cs stands for covalently-closed circular-synthesised, because the RNA elements used for CRISPR-Cas are generated with the help of a circular synthesis and are thus distributed more uniformly. With a whole library of such RNA rings, any gene in a cell can be specifically addressed in order to change it or switch it off.

They write: “3Cs multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage.”

The new 3Cs multiplex technique now even allows the simultaneous manipulation of two genes in one cell. Kaulich explains: “We can produce ‘address’ RNA libraries for all conceivable two-gene combinations. This allows up to several million combinations to be tested simultaneously in one experiment.”

Using the example of various genes involved in degradation processes, the research group demonstrated the potential of the new 3Cs multiplex technique: they examined almost 13,000 two-way combinations of genes that are responsible for recycling processes (autophagy) in the cell. With their help, the cell breaks down and recycles “worn-out” cell components. Disturbances in autophagy can trigger cell proliferation.

“Using the 3Cs multiplex technique, we were able to identify, for example, two genes involved in autophagy whose switching off leads to an uncontrolled growth of cells,” explains Kaulich. “These are precisely the autophagy mutations that occur in every fifth patient with squamous cell carcinoma of the lung. In this way, we can search very efficiently in cell culture experiments for genes that play an important role in cancer, and also in diseases of the nervous and immune systems, and that are suitable as possible targets for therapies.”

In that screen, the team identified synthetic lethal WDR45B-PIK3R4 and the proliferation-enhancing ATG7-KEAP1 genetic interactions. In the reporter-based screen, they found over 1,570 essential genetic interactions for autophagy flux, including interactions among paralogous genes, namely ATG2A-ATG2B, GABARAP-MAP1LC3B and GABARAP-GABARAPL2.