CRISPR-cas mediated silencing as an intelligent and iterative screening tool for bacterial engineering.

In its natural context, CRISPR-cas is a family of prokaryotic RNA-guided defence systems that direct degradation of invading viruses and “learn” from past infections to create a database of targets. Over the years, numerous tools have been developed that employ this custom-target cutting for genome editing, e.g. as a programmable restriction enzyme, or for specific gene shutdown when transcription is targeted. We intend to go further and develop a mechanism that harnesses the natural learning ability of CRISPR-cas systems to perform progressive silencing of multiple genes in a single experiment. This knowledge will serve as a basis for optimization of bacterial strains in industrial applications, by inducing multiple random but traceable silencing mutations and recording their performance in a context of interest.