06 Mar 2019 - 3Cs technology opens new horizons for production of highly complex CRISPR/Cas libraries.

As published today in eLife, a team around IBC2 Group Leader Manuel Kaulich has developed a new technology for generating CRISPR/Cas gene perturbation reagents. They dubbed their innovation as “3Cs”, as gRNA are synthesized as covalently-closed circles.

It enables the fast and, most importantly, cloning-free production of gRNA and shRNA libraries. By this, the 3Cs technology overcomes one of the major caveats of conventional library production.

Another advantage is that the 3Cs technology uncouples sequence diversity from sequence distribution, thereby removing an often-observed bias in the production process and rendering high quality reagents in any order of magnitude.

Groundbreaking discoveries can hardly ever be planned, and this one came over a coffee break. Manuel and Andreas Ernst, who at that time also headed an IBC2 group, were talking about their different areas of expertise, and suddenly the idea emerged on how to elegantly combine strategies used in phage display with gene editing. With support of Innovectis GmbH, Goethe University’s Tech Transfer Office, they immediately applied for patent protection.

To prove performance of 3Cs gene editing reagents in cells, together with the group of IBC2 Group Leader Anja Bremm a library targeting all human deubiquitinating enzymes (DUBs) was produced and their function for cell fitness identified. Then, the team around Manuel went on to generate the largest gRNA library known to date.

This so-called oTGW (optimized truly genome wide) library targets 16.5 million unique sites throughout the human genome, both in coding and non-coding regions. The oTGW library has a great potential to fuel future functional genomics studies and is available for the scientific community via the Goethe University Depository. Together with IBC2 Director Ivan Dikic, Manuel set up the Frankfurt CRISPR/Cas Screening Center (FCSC)