standard Repurposing CRISPR/Cas9 System to Reprogram Gene Expression

Sanyuan Ma

Sanyuan Ma, Ph.D. State Key Laboratory of Silkworm Genome Biology, Southwest University. Chongqing, P. R. China
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Zalatan et al. in Cell reported a system to generate synthetic transcriptional programs in yeast and human cells based on modified CRISPR-associated RNA scaffolds that could also be used in insects.

To fight against invading viruses and plasmids, bacteria developed multiple defense systems, one of which is the clustered regularly interspaced short palindromic repeat (CRISPR). CRISPR is an adaptive immune system consisting of genetic loci that can take up genetic material from invaders and later use it to target and interfere with the genetic information of returning invaders.

Cas9 with functional domains highlighted in relation to target DNA and guideRNA

Cas9 with functional domains highlighted in relation to target DNA and guideRNA

In recent years, the type II CRISPR system, CRISPR/Cas9, has been engineered to be an efficient genome editing tool. Together with zinc finger nucleases (ZFN) and transcription acitivation-like effector nucleases (TALEN), CRISPR/Cas9 system is pushing biological research into a new era in which site-directed manipulation of DNA is almost routine.

There are sophisticated transcription networks in eukaryotic cells and the ability to manipulate these networks will greatly help to elucidate the mechanisms underlying specific cell functions, and may provide powerful tools to the design cells and organisms with new functions.

Others have reported the use of modified Cas9 proteins onto which activation- or repressor-protein domains have been added, enabling the creation of customized programmable transcription factors.

scRNA

Cas9 can be modified to act as a repressor or activator. Zaltan et al. add specific protein binding domains to guide RNAs that encode ‘repressor’ or ‘activator’ binding information, allowing for more complex multilocus transcriptional regulation. From Zalatan et al.

Zalatan et al. have taken a different approach and have modified the small guide RNA that directs Cas9 to target locations to also contain RNA motifs that specifically bind proteins. They call these modified guide RNAs ‘scaffold RNAs’ – scRNA.

By encoding ‘activation’ or ‘repression’ information into scRNAs Zalatan et al. have enabled both activities to be programmed in a single genome using the CRISPR system.

Using these scRNAs, they showed that dCas9 (disabled) could be guided to target genes, while at the same time engineered transcriptional activators or repressors could be recruited by interacting with the newly added RNA motifs on the guide RNA. thus enabling flexible and parallel programmable locus-specific regulation.

For the proof-of-principle demonstration, Zalatan et al. chose the budding yeast S. cerevisiae as a model organism and show that this strategy enable flexible and parallel programmable locus-specific regulation.

Three orthogonal RNA-binding modules were identified and scRNAs were designed and optimized.

When they tested this system to human cells their results were positive with both reporter genes and endogenous genes.

Furthermore, Zalatan et al. showed the great power of this system to construct synthetic multigene expression programs. As the authors showed in the last part of their paper, this new system could regulate several genes in a biosynthetic pathway to redirect the pathway between different possible output states.

This paper describes a new and powerful tool for researchers working on gene expression programing that will be generally applicable to any system in which the CRISPR/Cas9 sytem can be deployed and this includes a growing number of insect systems.

Zalatan, Jesse G., Lee, Michael E., Almeida, Ricardo, , Gilbert, Luke A., Whitehead, Evan H., La Russa, Marie, Tsai, Jordan C., Weissman, Jonathan S., Dueber, John E., Qi, Lei S., Lim, Wendell A. (2015) Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell: 160: 339-350. doi 10.1016/j.cell.2014.11.052

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