A Modular Cloning Toolkit for Genome Editing in Cereals

The CRISPR/Cas technology has recently become the tool of choice for gene editing applications in plants.Here we describe the method of assembling CRISPR/Cas DNA constructs, using the modular cloning (MoClo), for deployment in cereal species. For this purpose, we use a cloning toolkit that consists of a library of Golden Gate (GG) modules, which encode genetic elements, such as promoters, coding sequences of CRISPR/Cas nucleases (e.g. Cas9), terminators, guide RNA backbones, etc. A part of the toolkit are modulesthat allow assembly of tRNA-sgRNA polycistronic units without a PCR step involved. The tRNA-sgRNA system has proved to be efficient in a number of plant species, including cereals (rice, wheat). The cloning toolkit we present can be used for a variety of gene editing applications, including gene deletions and single nucleotide substitutions. It can also be easily expanded by adding new standardised parts.