New 3Cs multiplex technique based on CRISPR-Cas technology helps study cancer mutations

Cancer and many other diseases are based on genetic defects. The body can often compensate for a defect in a gene; only the combination of several genetic defects leads to the clinical picture.

The 3Cs multiplex method based on CRISPR-Cas technology, developed at Goethe University Frankfurt, now offers the possibility of simulating millions of such combinations of genetic defects and examining their effects in cell cultures. These “gene scissors” enable the targeted introduction, removal and deactivation of genes. For this purpose, small snippets of genetic material (“single guide RNA”) are used as “addresses” that guide the gene scissors to certain sections of the DNA, where the gene scissors then become active.

The scientists at the Institute for Biochemistry II at Goethe University have expanded the 3Cs technology they developed and patented three years ago. 3Cs stands for Covalently-Closed-Circular-Synthesed, as the RNA elements used for CRISPR-Cas are generated with the help of a circular synthesis and are thus distributed more evenly. With a whole library of such RNA rings, any gene in a cell can be targeted in order to change or switch it off.

The new 3Cs multiplex technology now even allows the simultaneous manipulation of two genes in one cell.

We can make ‘Adress’ RNA libraries for all conceivable two-gene combinations. This means that up to several million combinations can be tested simultaneously in one experiment. “

Dr. Manuel Kaulich, Geothe University Frankfurt

So far, the effort for such experiments has been very high; the new technology from the research group reduces them, including costs, by a factor of ten. Thanks to the new 3Cs multiplex technology, the team can produce the address libraries in a very uniform and high quality. “Due to the mediocre quality of the CRISPR-Cas libraries available so far, very large experiments always had to be carried out in order to statistically compensate for any errors,” says Kaulich.

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

“With the 3Cs multiplex technique, for example, we were able to identify two genes involved in autophagy, the deactivation of which leads to uncontrolled cell growth,” explains Kaulich. “These are precisely the autophagy mutations that occur in every fifth patient with squamous cell carcinoma of the lungs. In cell culture experiments we can search very efficiently for genes that are used in cancer, but also in diseases of the nervous and immune systems, and which are suitable as possible targets for therapies. “

Source:

Journal reference:

Diehl, V., et al. (2021) Minimized combinatorial CRISPR screens identify genetic interactions in autophagy. Nucleic acid research. doi.org/10.1093/nar/gkab309.

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