CRISPR Cas9 Synthetic Eukaryotic Transcription Factors

Title:

Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas

Author(s):

Fahim Farzadfard Samuel D. Perli Timothy K. Lu

Publication:

Published in: ACS Synthetic Biology
Volume: 2, Issue: 10, Pages: 604-613
Published: 10/18/2013
DOI: 10.1021/sb400081r
Website: http://pubs.acs.org/doi/abs/10.1021/sb400081r

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The CRISPR Cas9 technology is used to design synthetic transcription factors in eukaryotes. The approach is used to design activators and repressors. Data shows that this approach can work in yeast and mammalian cells. This paper comes from Tim Lu’s group at MIT. A couple of years ago, we wrote a GenoCAD grammar to represent the structure of these synthetic transcription factors that was also published in ACS Synthetic Biology.

Transcriptional regulation is central to the complex behavior of natural biological systems and synthetic gene circuits. Platforms for the scalable, tunable, and simple modulation of transcription would enable new abilities to study natural systems and implement artificial capabilities in living cells. Previous approaches to synthetic transcriptional regulation have relied on engineering DNA-binding proteins, which necessitate multistep processes for construction and optimization of function. The authors show that the CRISPR Cas9 system ofStreptococcus pyogenes can be programmed to direct both activation and repression to natural and artificial eukaryotic promoters through the simple engineering of guide RNAs with base-pairing complementarity to target DNA sites. They demonstrate that the activity of CRISPR-based transcription factors (crisprTFs) can be tuned by directing multiple crisprTFs to different positions in natural promoters and by arraying multiple crisprTF-binding sites in the context of synthetic promoters in yeast and human cells. Furthermore, externally controllable regulatory modules can be engineered by layering gRNAs with small molecule-responsive proteins. Additionally, single nucleotide substitutions within promoters are sufficient to render them orthogonal with respect to the same gRNA-guided crisprTF. They envision that CRISPR Cas9 eukaryotic gene regulation will enable the facile construction of scalable synthetic gene circuits and open up new approaches for mapping natural gene networks and their effects on complex cellular phenotypes.