Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks
Genetic circuits require many regulatory parts in order to implement signal processing or execute algorithms in cells. A potentially scalable approach is to use dCas9, which employs small guide RNAs (sgRNAs) to repress genetic loci via the programmability of RNA:DNA base pairing. To this end, we use...
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
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Nature Publishing Group
2014
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| Online Access: | http://hdl.handle.net/1721.1/92529 https://orcid.org/0000-0003-0844-4776 https://orcid.org/0000-0003-2171-8460 |
| _version_ | 1811083318865690624 |
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| author | Nielsen, Alec Andrew Voigt, Christopher A. |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Nielsen, Alec Andrew Voigt, Christopher A. |
| author_sort | Nielsen, Alec Andrew |
| collection | MIT |
| description | Genetic circuits require many regulatory parts in order to implement signal processing or execute algorithms in cells. A potentially scalable approach is to use dCas9, which employs small guide RNAs (sgRNAs) to repress genetic loci via the programmability of RNA:DNA base pairing. To this end, we use dCas9 and designed sgRNAs to build transcriptional logic gates and connect them to perform computation in living cells. We constructed a set of NOT gates by designing five synthetic Escherichia coli σ[subscript 70] promoters that are repressed by corresponding sgRNAs, and these interactions do not exhibit crosstalk between each other. These sgRNAs exhibit high on‐target repression (56‐ to 440‐fold) and negligible off‐target interactions (< 1.3‐fold). These gates were connected to build larger circuits, including the Boolean‐complete NOR gate and a 3‐gate circuit consisting of four layered sgRNAs. The synthetic circuits were connected to the native E. coli regulatory network by designing output sgRNAs to target an E. coli transcription factor (malT). This converts the output of a synthetic circuit to a switch in cellular phenotype (sugar utilization, chemotaxis, phage resistance). |
| first_indexed | 2024-09-23T12:31:10Z |
| format | Article |
| id | mit-1721.1/92529 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T12:31:10Z |
| publishDate | 2014 |
| publisher | Nature Publishing Group |
| record_format | dspace |
| spelling | mit-1721.1/925292022-10-01T09:28:33Z Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks Nielsen, Alec Andrew Voigt, Christopher A. Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Synthetic Biology Center Nielsen, Alec Andrew Voigt, Christopher A. Genetic circuits require many regulatory parts in order to implement signal processing or execute algorithms in cells. A potentially scalable approach is to use dCas9, which employs small guide RNAs (sgRNAs) to repress genetic loci via the programmability of RNA:DNA base pairing. To this end, we use dCas9 and designed sgRNAs to build transcriptional logic gates and connect them to perform computation in living cells. We constructed a set of NOT gates by designing five synthetic Escherichia coli σ[subscript 70] promoters that are repressed by corresponding sgRNAs, and these interactions do not exhibit crosstalk between each other. These sgRNAs exhibit high on‐target repression (56‐ to 440‐fold) and negligible off‐target interactions (< 1.3‐fold). These gates were connected to build larger circuits, including the Boolean‐complete NOR gate and a 3‐gate circuit consisting of four layered sgRNAs. The synthetic circuits were connected to the native E. coli regulatory network by designing output sgRNAs to target an E. coli transcription factor (malT). This converts the output of a synthetic circuit to a switch in cellular phenotype (sugar utilization, chemotaxis, phage resistance). United States. Defense Advanced Research Projects Agency (CLIO N66001‐12‐C‐4016) National Institutes of Health (U.S.) (GM095765) National Institute of General Medical Sciences (U.S.) (Grant P50 GMO98792) Synthetic Biology Engineering Research Center (EEC0540879) United States. Defense Advanced Research Projects Agency (Ginkgo BioWorks. CLIO N66001‐12‐C‐4018) United States. Office of Naval Research. Multidisciplinary University Research Initiative (Grant N00014‐13‐1‐0074) United States. Office of Naval Research. Multidisciplinary University Research Initiative (Boston University. Award 4500000552) United States. Air Force Office of Scientific Research (FA9550‐11‐C‐0028) American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship (32 CFR 168a) 2014-12-29T18:43:53Z 2014-12-29T18:43:53Z 2014-11 Article http://purl.org/eprint/type/JournalArticle 1744-4292 http://hdl.handle.net/1721.1/92529 Nielsen, Alec A.K., and Christopher A. Voigt. “Multi-Input CRISPR/Cas Genetic Circuits That Interface Host Regulatory Networks.” Molecular Systems Biology 10, no. 11 (November 1, 2014): 763–763. https://orcid.org/0000-0003-0844-4776 https://orcid.org/0000-0003-2171-8460 en_US http://dx.doi.org/10.15252/msb.20145735 Molecular Systems Biology Creative Commons Attribution http://creativecommons.org/licenses/by/4.0/ application/pdf Nature Publishing Group EMBO Press |
| spellingShingle | Nielsen, Alec Andrew Voigt, Christopher A. Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks |
| title | Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks |
| title_full | Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks |
| title_fullStr | Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks |
| title_full_unstemmed | Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks |
| title_short | Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks |
| title_sort | multi input crispr cas genetic circuits that interface host regulatory networks |
| url | http://hdl.handle.net/1721.1/92529 https://orcid.org/0000-0003-0844-4776 https://orcid.org/0000-0003-2171-8460 |
| work_keys_str_mv | AT nielsenalecandrew multiinputcrisprcasgeneticcircuitsthatinterfacehostregulatorynetworks AT voigtchristophera multiinputcrisprcasgeneticcircuitsthatinterfacehostregulatorynetworks |