New Technique Optimizes Generation of Combinatorial CRISPR Libraries

Scientists from the Institute of Biochemistry II at Goethe University have expanded the 3Cs (covalently-closed circular-synthesized) technique that they developed and patented three years ago. The new 3Cs multiplex technique allows the simultaneous manipulation of two genes in one cell.

According to Manuel Kaulich, senior author of a paper published in Nucleic Acids Research last month, "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."

Until now, the cost and effort of such experiments was very high. The group's new technique reduces it, by a factor of ten, they say. This is because the team can produce the address libraries very uniformly and in high quality thanks to the 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 Kaulich.

Using the example of various genes involved in degradation processes, the group demonstrated the potential of the new 3Cs multiplex technique: they examined almost 13,000 two-way combinations of genes that are responsible for autophagy in the cell.

"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."