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

Abstract

SUMMARYIt 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 histonemodifying 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.

Genome Editing in Plants: An Overview of Tools and Applications

“…In addition, these technologies can be successfully used in the work on epigenome editing via the selection of proteins responsible for histone modification and DNA methylation, which has emerged as a new way of regulating cellular functions in plants [25]. For the purpose of understanding epigenetic regulation, CRISPR-Cas9 system can also be used for the enrichment of chromatin target sites for the identification of proteins attached to enriched chromatin.…”

CRISPR/Cas9-Mediated Genome Nano-Surgery: An Update

“…We can also alter gene expression by using a dead or broken Cas9 enzyme to block the binding of RNA polymerase needed for the gene to be expressed or attaching a dead or broken Cas9 protein to transcriptional factors or activator to stimulate the gene expression [29,30]. Furthermore, CRISPRCas9 was used to construct somatic and germline mouse models with point mutations or chromosomal deletions in multiple genes using multiple gRNAs, and even more complex chromosomal rearrangements.…”

“…Unlike conventional RNA interference, which cleaves only dsRNA using Dicer enzyme with high off-target effects and low reproducibility [34], reversible CRISPR/Cas9-mediated transcriptional repression can target any specific region throughout the whole genome with more predictable offtarget effect [35][36][37][38]. In the field of epigenetics, a dead Cas9 could be attached to epigenetic modifiers to activate DNA methylation by adding methyl group to DNA or to modify histones by adding acetyl groups to histone proteins, making epigenome reprogrammable and furthering our understanding of how a specific modification influences gene expression [30].…”

New developments in green biotechnology - an inventory for RIVM

“…Finally, a complex can be engineered to modify DNA epigenetically (e.g. by adding DNA methylating activity, see RNAi-TGS section 4.7, to the dCas9) in order to alter gene expression genome-wide (Puchta 2016;Thakore et al 2016). Very recently, Komor et al (2016) added another variant, i.e.…”

“…There is only one product authorised at present, against an orphan disease (Gaucher). Finally, engineered Cas9 without nuclease activity (dCas9) can be used to build novel transcription factors or epigenetic modifiers (Puchta 2016). Whether this remains a research tool or will be used in transgenic commercial applications, remains to be seen.…”