The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology.

Since the discovery that nucleases of the bacterial CRISPR/Cas system can be used as easily programmable tools for genome engineering, their application massively transformed different areas of plant biology. In this review, we assess the current state of their use for crop breeding to incorporate attractive new agronomical traits into specific cultivars of various crop plants. This can be achieved by the use of Cas9/12 nucleases for double strand break induction, resulting in mutations by non-homologous recombination. Strategies for performing such experiments - from the design of guide RNA to the use of different transformation technologies - are evaluated. Furthermore, we sum up recent developments regarding the use of nuclease-deficient Cas9/12 proteins, as DNA-binding moieties for targeting different kinds of enzyme activities to specific sites within the genome. Progress in base deamination, transcriptional induction and transcriptional repression, as well as in imaging in plants, is also discussed. As different Cas9/12 enzymes are at hand, the simultaneous application of various enzyme activities, to multiple genomic sites, is now in reach to redirect plant metabolism in a multifunctional manner and pave the way for a new level of plant synthetic biology.