Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas
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 cel...
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
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American Chemical Society (ACS)
2013
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| Online Access: | http://hdl.handle.net/1721.1/82091 https://orcid.org/0000-0002-8346-2184 https://orcid.org/0000-0003-1389-8203 https://orcid.org/0000-0002-9999-6690 |
| _version_ | 1811075353431506944 |
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| author | Farzadfard, Fahim Perli, Samuel David Lu, Timothy K |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Farzadfard, Fahim Perli, Samuel David Lu, Timothy K |
| author_sort | Farzadfard, Fahim |
| collection | MIT |
| description | 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. Here, we show that the CRISPR/Cas system of Streptococcus 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. We 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. We envision that CRISPR-based 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. |
| first_indexed | 2024-09-23T10:04:42Z |
| format | Article |
| id | mit-1721.1/82091 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T10:04:42Z |
| publishDate | 2013 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/820912022-09-30T18:44:38Z Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas Farzadfard, Fahim Perli, Samuel David Lu, Timothy K Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science Massachusetts Institute of Technology. Synthetic Biology Center Farzadfard, Fahim Perli, Samuel David Lu, Timothy K. 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. Here, we show that the CRISPR/Cas system of Streptococcus 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. We 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. We envision that CRISPR-based 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. United States. Defense Advanced Research Projects Agency National Institutes of Health (U.S.) (New Innovator Award 1DP2OD008435) National Science Foundation (U.S.) (1124247) 2013-11-12T17:19:24Z 2013-11-12T17:19:24Z 2013-08 2013-07 Article http://purl.org/eprint/type/JournalArticle 2161-5063 http://hdl.handle.net/1721.1/82091 Farzadfard, Fahim, Samuel D. Perli, and Timothy K. Lu. “Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas.” ACS Synthetic Biology 2, no. 10 (October 18, 2013): 604-613. © 2013 American Chemical Society https://orcid.org/0000-0002-8346-2184 https://orcid.org/0000-0003-1389-8203 https://orcid.org/0000-0002-9999-6690 en_US http://dx.doi.org/10.1021/sb400081r ACS Synthetic Biology Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society (ACS) PMC |
| spellingShingle | Farzadfard, Fahim Perli, Samuel David Lu, Timothy K Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas |
| title | Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas |
| title_full | Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas |
| title_fullStr | Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas |
| title_full_unstemmed | Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas |
| title_short | Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas |
| title_sort | tunable and multifunctional eukaryotic transcription factors based on crispr cas |
| url | http://hdl.handle.net/1721.1/82091 https://orcid.org/0000-0002-8346-2184 https://orcid.org/0000-0003-1389-8203 https://orcid.org/0000-0002-9999-6690 |
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