Using CRISPR/Cas in three dimensions: towards synthetic plant genomes, transcriptomes and epigenomes.

It is possible to target individual sequence motives within genomes by using synthetic DNA-binding domains. This one-dimensional approach has been used successfully in plants to induce mutations or for the transcriptional regulation of single genes. When the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system was discovered, a tool became available allowing the extension of this approach from one to three dimensions and to construct at least partly synthetic entities on the genome, epigenome and transcriptome levels. The second dimension can be obtained by targeting the Cas9 protein to multiple unique genomic sites by applying multiple different single guiding (sg) RNAs, each defining a different DNA-binding site. Finally, the simultaneous use of phylogenetically different Cas9 proteins or sgRNAs that harbour different types of protein binding motives, allows for a third dimension of control. Thus, different types of enzyme activities - fused either to one type of Cas9 orthologue or to one type of RNA-binding domain specific to one type of sgRNA - can be targeted to multiple different genomic sites simultaneously. Thus, it should be possible to induce quantitatively different levels of expression of certain sets of genes and at the same time to repress other genes, redefining the nuclear transcriptome. Likewise, by the use of different types of histone-modifying and/or DNA (de)methylating activities, the epigenome of plants should be reprogrammable. On our way to synthetic plant genomes, the next steps will be to use complex genome engineering approaches within or between species borders to restructure and recombine natural or artificial chromosomes.