Team:Freiburg/Project/1

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Abstract

English

Gene Regulation – Multiple Targets – Activation – Optogenetics – Repression – Inducibility – Epigenetics

Just imagine there was a tool that combined all these aspects of lab work. A tool, that was able to rule several genes at once; a tool that allowed highly specific gene modulation via stimulus induction; a tool that would be a new approach to gene regulation.

This year’s Freiburg iGEM Team uses the prokaryotic CRISPR/Cas system to enable multiple endogenous gene regulation with minimal effort. The regulation is based on a protein-RNA-DNA interaction. Customizable RNAs function as a guide for our protein in order to target specific DNA sequences. By fusing effector domains to this protein, we aim at developing a tool for multiple and inducible gene activation and repression. Despite the system's prokaryotic origin, gene target sequences are adjustable for various organisms, offering a broad application variety of our tool. Due to its great potential, the CRISPR/Cas system has become of increasing importance in current research and can be implemented in a number of novel and interesting applications, such as gene therapy or tissue engineering.

Introduction

CRISPR/Cas

Hidden as an uncharacterized E. coli locus for more than 15 years [1], Barrangou et al. first described a previously unknown adaptive prokaryotic immune system [2]. Almost half of all Eubacteria and most Archaea take advantage of this defence mechanism. Thereby, invasive DNA can be specifically and efficiently cleaved, controlling phage DNA transduction, unselective uptake through natural transformation and horizontal gene transfer by conjugation [3].

This immune system‘s unique feature results from a complex machinery of highly-selective splicing proteins and recombinases, non-coding RNAs and repetitive DNA spacers which, in turn, encode different potential invador target sequences [4]. All of these components lie highly structured and in close vicinity to each other - mostly on single operons. Such loci were labeled as Clustered Regularly Interspaced Short Pallindromic Repeats - CRISPR - and differ widely among and within a great variety of subsystems in different species [5, 6]. These findings hold for definite sequence order, ribonucleoprotein composition and functional mechanisms of CRISPRs. It wasn‘t before 2012 [7] that CRISPR associated proteins - Cas - and CRISPR RNAs - crRNAs -, the system‘s two key driving components, have aroused greater interest of Synthetic Biologists. Thus, til date, some detailed structural and functional characteristics of these constituents yet remain to be elucidated.

[ILLUSTRATION: CRISPR Locus / Interactive Graphic]

Unlike Zinkfingers, TAL effectors or Meganucleases, Cas9 proteins direct DNA sequence specific adhesion by harnessing a unique crRNA scaffold. With ~ 20 nucleotides corresponding to the target element, Watson-Crick base pairing can be established between crRNAs and the desired DNA. By their short size, crRNAs are easy to order, insert and express from vector plasmids, containing an RNA-Polymerase III driving U6 promoter. So far, the only constraint for recognition between crRNA and target DNA is a protospacer adjacent motif - PAM -, located directly 3‘ of the protospacer locus and containing a NGG triplett. A second, trans-acting crRNA - tracrRNA - mediates pre-processing of the crRNA and indispensably enhances formation of the Cas-crRNA ribonucleoprotein complex [7].

[ILLUSTRATION: CRISPR/Cas Functioning Mechanism / Interactive Graphic]

CRISPR/Cas9 systems could, in the near future, commonly be used to target multiple spacers. Thereby, co-transfecting of standardized crRNA array plasmids with a Cas9 protein and tracrRNA encoding plasmid, might yield a powerful device for multiplex genome engineering [8, 9]. It opens up the possibility to deal with a wide range of yet unadressed scientific questions in the near future, including Systems Biology aspects and complex metabolic approaches. Other advantages cover monetary and logistic aspects, as the only component for modification lies indeed within the crRNA itself. In turn, this can be ordered as two corresponding forward and reverse primers - see Team Freiburg's easy crRNA Designing Tool. Financial, logistic and human ressources stay at a minimum. Accordingly, CRISPR/Cas9 systems have already been established for many model organisms, including Saccharomyces cerevisiae, Caenorhabditis elegans, Arabidopsis thaliana, Drosophila melanogaster, Danio rerio and Mus musculus.

Sources

(1) Ishino, Y., et al. (1987). Nucleotide Sequence of the iap Gene in Escherichia coli. Journal of Bacteriology 169, 5429-5433.

(2) Barrangou, R., et al. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709-1712.

(3) Marraffini, L., and Sontheimer, E. (2008). CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843-1845.

(4) Horvath, P., and Barrangou, R. (2010). CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167-170.

(5) Jansen, R., et al. (2002). Identification of genes that are associated with DNA repeats in prokaryotes. Molecular Microbiology 43, 1565-1575.

(6) Makarova, K., et al. (2011). Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9, 467-477.

(7) Jinek, M., et al. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821.

(8) Cong, L., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823.

(9) Mali, P., et al. (2013). RNA-guided human genome engineering via Cas9. Science 339, 823-826.

Results

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@@ Line 175: / Line 175: @@
 <div>
-<p>Hidden as an uncharacterized <i>E. coli</i> locus for more than 15 years<a href="#(1)">[1]</a>, Barrangou et al. first described a previously unknown adaptive prokaryotic immune system<a href="#(2)">[2]</a>. Almost half of all <i>Eubacteria</i> and most <i>Archaea</i> take advantage of this defence mechanism. Thereby, invasive DNA can be specifically and efficiently cleaved, controlling phage DNA transduction, unselective uptake through natural transformation and horizontal gene transfer by conjugation<a href="#(3)">[3]</a>.</p> <br>
+<p>Hidden as an uncharacterized <i>E. coli</i> locus for more than 15 years<a href="#(1)"> [1]</a>, Barrangou et al. first described a previously unknown adaptive prokaryotic immune system<a href="#(2)"> [2]</a>. Almost half of all <i>Eubacteria</i> and most <i>Archaea</i> take advantage of this defence mechanism. Thereby, invasive DNA can be specifically and efficiently cleaved, controlling phage DNA transduction, unselective uptake through natural transformation and horizontal gene transfer by conjugation<a href="#(3)"> [3]</a>.</p> <br>
-<p>This immune system‘s unique feature results from a complex machinery of highly-selective splicing proteins and recombinases, non-coding RNAs and repetitive DNA spacers which, in turn, encode different potential invador target sequences<a href="#(4)">[4]</a>. All of these components lie highly structured and in close vicinity to each other - mostly on single operons. Such loci were labeled as <b>C</b>lustered <b>R</b>egularly <b>I</b>nterspaced <b>S</b>hort <b>P</b>allindromic <b>R</b>epeats - CRISPR - and differ widely among and within a great variety of subsystems in different species<a href="#(5)">[5, 6]</a>. These findings hold for definite sequence order, ribonucleoprotein composition and functional mechanisms of CRISPRs. It wasn‘t before 2012<a href="#(7)">[7]</a> that <b>C</b>RISPR <b>a</b>ssociated <b>p</b>roteins - Cas - and <b>CR</b>ISPR <b>RNA</b>s - crRNAs -, the system‘s two key driving components, have aroused greater interest of Synthetic Biologists. Thus, til date, some detailed structural and functional characteristics of these constituents yet remain to be elucidated.</p><br></div>
+<p>This immune system‘s unique feature results from a complex machinery of highly-selective splicing proteins and recombinases, non-coding RNAs and repetitive DNA spacers which, in turn, encode different potential invador target sequences<a href="#(4)"> [4]</a>. All of these components lie highly structured and in close vicinity to each other - mostly on single operons. Such loci were labeled as <b>C</b>lustered <b>R</b>egularly <b>I</b>nterspaced <b>S</b>hort <b>P</b>allindromic <b>R</b>epeats - CRISPR - and differ widely among and within a great variety of subsystems in different species<a href="#(5)"> [5, 6]</a>. These findings hold for definite sequence order, ribonucleoprotein composition and functional mechanisms of CRISPRs. It wasn‘t before 2012<a href="#(7)"> [7]</a> that <b>C</b>RISPR <b>a</b>ssociated <b>p</b>roteins - Cas - and <b>CR</b>ISPR <b>RNA</b>s - crRNAs -, the system‘s two key driving components, have aroused greater interest of Synthetic Biologists. Thus, til date, some detailed structural and functional characteristics of these constituents yet remain to be elucidated.</p><br></div>
 <div align="center"><b>[ILLUSTRATION: CRISPR Locus / Interactive Graphic]</b><br><br></div>
 <div>
-<p>Unlike Zinkfingers, TAL effectors or Meganucleases, Cas9 proteins direct DNA sequence specific adhesion by harnessing a unique crRNA scaffold. With ~ 20 nucleotides corresponding to the target element, Watson-Crick base pairing can be established between crRNAs and the desired DNA. By their short size, crRNAs are easy to order, insert and express from vector plasmids, containing an RNA-Polymerase III driving U6 promoter. So far, the only constraint for recognition between crRNA and target DNA is a <b>p</b>rotospacer <b>a</b>djacent <b>m</b>otif - PAM -, located directly 3‘ of the protospacer locus and containing a NGG triplett. A second, <b>tra</b>ns-acting <b>cr</b>RNA - tracrRNA - mediates pre-processing of the crRNA and indispensably enhances formation of the Cas-crRNA ribonucleoprotein complex<a href="#(7)">[7]</a>.</p><br></div>
+<p>Unlike Zinkfingers, TAL effectors or Meganucleases, Cas9 proteins direct DNA sequence specific adhesion by harnessing a unique crRNA scaffold. With ~ 20 nucleotides corresponding to the target element, Watson-Crick base pairing can be established between crRNAs and the desired DNA. By their short size, crRNAs are easy to order, insert and express from vector plasmids, containing an RNA-Polymerase III driving U6 promoter. So far, the only constraint for recognition between crRNA and target DNA is a <b>p</b>rotospacer <b>a</b>djacent <b>m</b>otif - PAM -, located directly 3‘ of the protospacer locus and containing a NGG triplett. A second, <b>tra</b>ns-acting <b>cr</b>RNA - tracrRNA - mediates pre-processing of the crRNA and indispensably enhances formation of the Cas-crRNA ribonucleoprotein complex<a href="#(7)"> [7]</a>.</p><br></div>
 <div align="center"><b> [ILLUSTRATION: CRISPR/Cas Functioning Mechanism / Interactive Graphic]</b></div><br><br>
 <div>
-<p>CRISPR/Cas9 systems could, in the near future, commonly be used to target multiple spacers. Thereby, co-transfecting of standardized crRNA array plasmids with a Cas9 protein and tracrRNA encoding plasmid, might yield a powerful device for multiplex genome engineering<a href="#(8)">[8, 9]</a>. It opens up the possibility to deal with a wide range of yet unadressed scientific questions in the near future, including Systems Biology aspects and complex metabolic approaches. Other advantages cover monetary and logistic aspects, as the only component for modification lies indeed within the crRNA itself. In turn, this can be ordered as two corresponding forward and reverse primers - see Team Freiburg's easy <b><a href="https://2013.igem.org/Team:Freiburg/Project/crrna#design_tool"; LINK="white"; ALINK="white"; VLINK="white">crRNA Designing Tool</a></b>. Financial, logistic and human ressources stay at a minimum. Accordingly, CRISPR/Cas9 systems have already been established for many model organisms, including <i>Saccharomyces cerevisiae</i>, <i>Caenorhabditis elegans</i>, <i>Arabidopsis thaliana</i>, <i>Drosophila melanogaster</i>, <i>Danio rerio</i> and <i>Mus musculus</i>.</p><br>
+<p>CRISPR/Cas9 systems could, in the near future, commonly be used to target multiple spacers. Thereby, co-transfecting of standardized crRNA array plasmids with a Cas9 protein and tracrRNA encoding plasmid, might yield a powerful device for multiplex genome engineering<a href="#(8)"> [8, 9]</a>. It opens up the possibility to deal with a wide range of yet unadressed scientific questions in the near future, including Systems Biology aspects and complex metabolic approaches. Other advantages cover monetary and logistic aspects, as the only component for modification lies indeed within the crRNA itself. In turn, this can be ordered as two corresponding forward and reverse primers - see Team Freiburg's easy <b><a href="https://2013.igem.org/Team:Freiburg/Project/crrna#design_tool"; LINK="white"; ALINK="white"; VLINK="white">crRNA Designing Tool</a></b>. Financial, logistic and human ressources stay at a minimum. Accordingly, CRISPR/Cas9 systems have already been established for many model organisms, including <i>Saccharomyces cerevisiae</i>, <i>Caenorhabditis elegans</i>, <i>Arabidopsis thaliana</i>, <i>Drosophila melanogaster</i>, <i>Danio rerio</i> and <i>Mus musculus</i>.</p><br>
 <p><small><b>Sources</b> <br>