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Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. ​

Literature Information ​

DOI10.1093/nar/gkt520
PMID23761437
JournalNucleic acids research
Impact Factor13.1
JCR QuartileQ1
Publication Year2013
Times Cited539
KeywordsCRISPR-Cas system, gene expression regulation, transcription activation, transcription repression, synthetic biology
Literature TypeJournal Article, Research Support, N.I.H., Extramural
ISSN0305-1048
Pages7429-37
Issue41(15)
AuthorsDavid Bikard, Wenyan Jiang, Poulami Samai, Ann Hochschild, Feng Zhang, Luciano A Marraffini

TL;DR ​

This study presents a novel approach to controlling gene transcription using a Cas9 nuclease mutant, which can be engineered to function as a programmable transcription repressor or activator. This technology offers a simple and efficient method for modulating gene expression, significantly advancing research in gene networks and synthetic biology applications.

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CRISPR-Cas system · gene expression regulation · transcription activation · transcription repression · synthetic biology

Abstract ​

The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.

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Primary Questions Addressed ​

  1. What are the potential applications of programmable transcription repression and activation in synthetic biology?
  2. How does the engineered CRISPR-Cas system compare to traditional methods of gene expression control?
  3. What specific challenges might arise when implementing this technology in different bacterial species?
  4. Can this CRISPR-Cas system be adapted for use in eukaryotic organisms, and what modifications would be necessary?
  5. What are the implications of this technology for understanding complex gene regulatory networks in bacteria?

Key Findings ​

Research Background and Purpose ​

The study explores the development of an engineered CRISPR-Cas system for the programmable repression and activation of bacterial gene expression. The primary goal is to provide a versatile tool for controlling transcription in prokaryotes, which is essential for studying gene function and creating synthetic gene networks.

Main Methods/Materials/Experimental Design ​

The authors utilized a mutant form of the Cas9 nuclease, termed "dead" Cas9 (dCas9), which retains DNA-binding capabilities but lacks nuclease activity. The study involved several key experimental approaches:

  1. Strains and Culture Conditions: E. coli and Streptococcus pneumoniae were used as model organisms, with specific antibiotics for selection.
  2. Plasmid Construction: Various plasmids were created to express crRNA guides and dCas9, allowing targeted gene regulation.
  3. Transcription Repression and Activation: dCas9 was directed to promoter regions to inhibit transcription initiation or to open reading frames to block elongation. The fusion of dCas9 with the omega subunit of RNA polymerase (RNAP) was tested for transcription activation.
  4. Fluorescence Measurements: GFP fluorescence assays and β-galactosidase activity measurements were used to quantify gene expression levels.
  5. Northern Blot Analysis: This was employed to confirm transcription levels and the effects of dCas9 binding on RNA production.
Mermaid diagram

Key Results and Findings ​

  1. Transcription Repression: dCas9 effectively repressed gene expression in both E. coli and S. pneumoniae, with up to a 100-fold reduction in fluorescence observed when directed to promoter regions.
  2. Transcription Activation: The fusion of dCas9 with the RNAP omega subunit resulted in significant activation of gene expression, achieving up to 23-fold induction in some cases.
  3. Mismatches in crRNA: Introducing mismatches in crRNA allowed for modulation of repression levels, demonstrating that even partial complementarity could lead to gene regulation.

Main Conclusions/Significance/Innovativeness ​

The study presents a powerful method for the precise control of gene expression in bacteria using an engineered CRISPR-Cas system. This technology simplifies the manipulation of transcription without requiring modifications to the target gene's promoter. The ability to both repress and activate transcription provides a versatile platform for synthetic biology applications, enabling researchers to construct complex genetic circuits and study gene networks more effectively.

Research Limitations and Future Directions ​

  • Limitations: The study primarily focused on E. coli and S. pneumoniae, which may limit the applicability of the findings to other bacterial species. Additionally, potential off-target effects of dCas9 binding need further investigation.
  • Future Directions: Future research could explore the application of this technology in diverse bacterial systems, optimize the design of crRNA for improved specificity, and investigate the use of dCas9 in eukaryotic cells. Additionally, the potential for multiplexing with multiple crRNA guides opens avenues for more complex genetic manipulations.

Summary Table of Key Findings ​

AspectFindings
Repression EfficiencyUp to 100-fold reduction in gene expression
Activation EfficiencyUp to 23-fold induction of target genes
Modulation via MismatchesPartial complementarity allowed for fine-tuning of expression levels
Application PotentialUseful for synthetic biology and genetic circuit design

References ​

  1. Gene-expression tools for the metabolic engineering of bacteria. - J D Keasling - Trends in biotechnology (1999)
  2. Efficient genome editing in zebrafish using a CRISPR-Cas system. - Woong Y Hwang;Yanfang Fu;Deepak Reyon;Morgan L Maeder;Shengdar Q Tsai;Jeffry D Sander;Randall T Peterson;J-R Joanna Yeh;J Keith Joung - Nature biotechnology (2013)
  3. Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site. - Asma Hatoum-Aslan;Inbal Maniv;Luciano A Marraffini - Proceedings of the National Academy of Sciences of the United States of America (2011)
  4. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. - Hélène Deveau;Rodolphe Barrangou;Josiane E Garneau;Jessica Labonté;Christophe Fremaux;Patrick Boyaval;Dennis A Romero;Philippe Horvath;Sylvain Moineau - Journal of bacteriology (2008)
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  6. Unlocking the potential of the human genome with RNA interference. - Gregory J Hannon;John J Rossi - Nature (2004)
  7. FACS-optimized mutants of the green fluorescent protein (GFP). - B P Cormack;R H Valdivia;S Falkow - Gene (1996)
  8. Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. - Le Cong;Ruhong Zhou;Yu-Chi Kuo;Margaret Cunniff;Feng Zhang - Nature communications (2012)
  9. A synthetic biology framework for programming eukaryotic transcription functions. - Ahmad S Khalil;Timothy K Lu;Caleb J Bashor;Cherie L Ramirez;Nora C Pyenson;J Keith Joung;James J Collins - Cell (2012)
  10. Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa. - Michael E Zegans;Jeffrey C Wagner;Kyle C Cady;Daniel M Murphy;John H Hammond;George A O'Toole - Journal of bacteriology (2009)

Literatures Citing This Work ​

  1. Optical control of mammalian endogenous transcription and epigenetic states. - Silvana Konermann;Mark D Brigham;Alexandro Trevino;Patrick D Hsu;Matthias Heidenreich;Le Cong;Randall J Platt;David A Scott;George M Church;Feng Zhang - Nature (2013)
  2. CRISPR RNA-guided activation of endogenous human genes. - Morgan L Maeder;Samantha J Linder;Vincent M Cascio;Yanfang Fu;Quan H Ho;J Keith Joung - Nature methods (2013)
  3. A ruler protein in a complex for antiviral defense determines the length of small interfering CRISPR RNAs. - Asma Hatoum-Aslan;Poulami Samai;Inbal Maniv;Wenyan Jiang;Luciano A Marraffini - The Journal of biological chemistry (2013)
  4. Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. - Fahim Farzadfard;Samuel D Perli;Timothy K Lu - ACS synthetic biology (2013)
  5. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. - Kevin M Esvelt;Prashant Mali;Jonathan L Braff;Mark Moosburner;Stephanie J Yaung;George M Church - Nature methods (2013)
  6. Cas9 as a versatile tool for engineering biology. - Prashant Mali;Kevin M Esvelt;George M Church - Nature methods (2013)
  7. Chromosomal targeting by CRISPR-Cas systems can contribute to genome plasticity in bacteria. - Ron L Dy;Andrew R Pitman;Peter C Fineran - Mobile genetic elements (2013)
  8. Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors. - Andrew C Mercer;Thomas Gaj;Shannon J Sirk;Brian M Lamb;Carlos F Barbas - ACS synthetic biology (2014)
  9. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. - Ines Fonfara;Anaïs Le Rhun;Krzysztof Chylinski;Kira S Makarova;Anne-Laure Lécrivain;Janek Bzdrenga;Eugene V Koonin;Emmanuelle Charpentier - Nucleic acids research (2014)
  10. Control of gene expression by CRISPR-Cas systems. - David Bikard;Luciano A Marraffini - F1000prime reports (2013)

... (529 more literatures)

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