Team:Freiburg/Project/1

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<p class="second_order"> <a href="#introduction"> Introduction </a> </p>
<p class="second_order"> <a href="#introduction"> Introduction </a> </p>
<p class="second_order"> <a href="#background"> CRISPR/Cas</a> </p>
<p class="second_order"> <a href="#background"> CRISPR/Cas</a> </p>
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<p class="second_order"> <a href="#outlook"> Outlook</a> </p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/crrna"> Targeting </a></p>
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/effector"> Effectors </a></p>
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/effector"> Effectors </a></p>
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/induction"> Effector Control </a> </p>
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/induction"> Effector Control </a> </p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/crrna"> Targeting </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/modeling"> Modeling </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/truncation"> Truncation </a></p>
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/method"> uniBAss </a></p>
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/method"> uniBAss </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/unibox"> uniBOX </a></p>
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/toolkit"> Manual </a></p>
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/toolkit"> Manual </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/modeling"> Modeling </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/application" > Application </a></p>
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Our Team developed a universal toolkit, termed uniCAS, that enables customizable gene regulation in mammalian cells. Therefore, we engineered the recently understood and highly promising CRISPR/Cas9 system. The regulation is based on the RNA-guided Cas9 protein, which allows specific targeting of DNA sequences. Our toolkit comprises not only a standardized Cas9 protein, but also different effector domains for efficient gene activation or repression. We further engineered a modular RNA plasmid for easy implementation of RNA guide sequences. As an additional feature, we established an innovative screening method for assessing the functionality of our uniCAS fusion proteins. Single genes and even whole genetic networks can be modified using our uniCAS toolkit. We think that our toolbox of standardized parts of the CRISPR/Cas9 system offers broad application in research fields such as tissue engineering, stem cell reprogramming and fundamental research.
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Our team developed a universal toolkit, termed uniCAS, that enables customizable gene regulation in mammalian cells. Therefore, we engineered the recently understood and highly promising CRISPR/Cas9 system. The regulation is based on the RNA-guided Cas9 protein, which allows specific targeting of DNA sequences. Our toolkit comprises not only a standardized Cas9 protein, but also different effector domains for efficient gene activation or repression. We further engineered a modular RNA plasmid for easy implementation of RNA guide sequences. As an additional feature, we established an innovative screening method for assessing the functionality of our uniCAS fusion proteins. Single genes and even whole genetic networks can be modified using our uniCAS toolkit. We think that our toolbox of standardized parts of the CRISPR/Cas9 system offers broad application in research fields such as tissue engineering, stem cell reprogramming and fundamental research.
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<a id="link" href="https://2013.igem.org/Team:Freiburg/description"> Click here for a multilingual project description </a>
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Introduction to our project
Introduction to our project
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<p>The enormous amount of gene regulation, signal transduction and metabolic pathways gives us a slight idea about what complexity of life really means. This astonishing complexity is one of the most crucial issues to understand, if we want to understand life itself.</p>
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<p>The enormous amount of gene regulation, signal transduction and metabolic pathways gives us a slight idea about what complexity of life really means. This astonishing complexity is one of the most crucial issues to understand, if we want to understand life itself.</p><!--if we want to understand this astonishing phenomenon, one approach is to attempt deciphering nature's elaborate gene networks with . -->
<p>Many approaches have been investigated and given us insights into these biological pathways, resulting in more and more elaborate tools. Solutions for the treatment of systemic diseases like cancer or the fine-tuning of cellular processes remain challenging. There is a need for a powerful tool, which enables powerful regulation of whole genetic networks and yet is simple to use. In the last years there have been several approaches which were able to alter gene expression, but they have the flaw that they are only targeting one gene at a time and do not allow flexible modification of entire genomes.</p>
<p>Many approaches have been investigated and given us insights into these biological pathways, resulting in more and more elaborate tools. Solutions for the treatment of systemic diseases like cancer or the fine-tuning of cellular processes remain challenging. There is a need for a powerful tool, which enables powerful regulation of whole genetic networks and yet is simple to use. In the last years there have been several approaches which were able to alter gene expression, but they have the flaw that they are only targeting one gene at a time and do not allow flexible modification of entire genomes.</p>
<p>In the last 8 months, our team has been planning, developing and building up a universal toolkit, which enables the regulation of complex networks. A toolkit that allows the simple and flexible regulation of multiple genes by only one protein.</p>
<p>In the last 8 months, our team has been planning, developing and building up a universal toolkit, which enables the regulation of complex networks. A toolkit that allows the simple and flexible regulation of multiple genes by only one protein.</p>
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<td> <b>Figure 1: Our uniCAS toolkit </b><br>
<td> <b>Figure 1: Our uniCAS toolkit </b><br>
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The uniCAS toolkit provides 3 different <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/effector">effectors</a>, 2 methods & 1 <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/induction">effector controller</a>! Using our toolkit it's possible to efficiently activate or repress genes. We also provide devices for effector controling by light. For multiple targeting different targets can be easily combined using the <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/crrna#rnaimer">RNAimer</a> plasmid.<br>
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The uniCAS toolkit provides 3 different <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/effector">effectors</a>, 2 methods & 1 <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/induction">effector control</a>! By using our toolkit it is possible to efficiently activate or repress gene expression. We also provide devices for controlling our effectors by light. For multiple targeting different DNA sites can be addressed simultaneously by using our <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/crrna#rnaimer">RNAimer</a> plasmid.<br>
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Use our custom-tailored <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/toolkit">Manual Tool</a> to generate your individual <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/toolkit">manual</a> for your needs of gene regulation.
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Try working with uniCAS and receive custom-tailored <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/toolkit">instructions</a> for your desired gene regulation experiment by using our <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/toolkit">Manual Tool</a>.
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<p><br></p>
<p><br></p>
<p>The great potential harbored by the <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/1#background"> CRISPR/Cas9 </a> system provided a great basis for us to modify it in a way that allows multiple, sequence specific gene regulation in mammalian cells.<br>
<p>The great potential harbored by the <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/1#background"> CRISPR/Cas9 </a> system provided a great basis for us to modify it in a way that allows multiple, sequence specific gene regulation in mammalian cells.<br>
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The first step to a universal toolkit was to generate a sequence specific DNA-binding protein. This was archieved by mutating the DNA-cleavage site of the Cas9 herwith termed dCas9 protein. With the catalytically inactive engineered mutant of Cas9 we were able to influence the DNA-binding locus and direct the protein to requested DNA targets.  
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To establish a tool that influences the gene expression in mammalian cells, we fused different effectors to the dCas9 protein. For gene regulation we used the trans-activator <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/effector#activation">VP16</a>, the repressor-domain <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/effector#repression">KRAB</a> and the histone methyltransferase <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/effector#epigenetics">G9a</a>. These effectors were able to efficiently change the expression rate of a reporter protein SEAP (secreted alcaline phosphatase) and the endogenous signal protein VEGF (vascular endothelial growth factor).</p>
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<p style=" border:thin solid  white; margin:15px; padding:15px;"><b>The CRISPR/Cas9 system in short</b>:<br>
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This CRISPR/Cas9 system is based on the interaction of three acting components: tracrRNA, crRNA and Cas9 protein. <!-- <span id="refer"> <a href="#(7)"> [7]</a></span>. --> The crRNA detects complementary invasive DNA, tracrRNA enables the interaction of crRNA and Cas9. The RNA-Cas9 complex binds respective DNA and Cas9 cleaves the DNA sequcence specifically.</p>
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<br>
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<!--<p id="h4"> The engineered system </p>-->
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<p>We used a <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/attributions#Zhang">human codon optimized Cas9</a>, mutated the remaining nickase function and standardized the 160 kDa protein. The result: The DNA binding protein dCas9 which can be guided specifically to any DNA target site due to complexation with crRNA (CRISPR RNA) and tracrRNA (transactivating crRNA). By fusing different effector domains to dCas9 we built fusion proteins for gene regulation in mammalian cells.</p>
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<td> <b>Figure 3: Engineering of the CRISPR/Cas9 system </b><br>
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<td> <b>Figure 2: Engineering of the CRISPR/Cas9 system </b><br>
The nuclease function of Cas9 has been mutated. The dCas9 is still able to bind the guiding RNAs and therewith can be directed towards every requested sequence. By fusing effectors to the dCas9, gene regulation of specitic loci can be engineered.
The nuclease function of Cas9 has been mutated. The dCas9 is still able to bind the guiding RNAs and therewith can be directed towards every requested sequence. By fusing effectors to the dCas9, gene regulation of specitic loci can be engineered.
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<p><br></p>
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<!-- The first step to a universal toolkit was to generate a sequence specific DNA-binding protein. This was achieved by mutating the DNA-cleavage site of the Cas9 herewith termed dCas9 protein. With the catalytically inactive engineered mutant of Cas9 we were able to influence the DNA-binding locus and direct the protein to requested DNA targets.
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To establish a tool that influences the gene expression in mammalian cells, we fused different effectors to the dCas9 protein.-->  
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<br>
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For gene regulation we used the trans-activator <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/effector#activation">VP16</a>, the repressor-domain <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/effector#repression">KRAB</a> and the histone methyltransferase <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/effector#epigenetics">G9a</a>. These effectors were able to efficiently change the expression rate of a reporter protein SEAP (secreted alcaline phosphatase) and the endogenous signal protein VEGF (vascular endothelial growth factor).</p>
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<p>In order to allow for control of gene regulation in a time- and space-dependent manner, we designed various control elements. We engineered and tested <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/induction#light">light induction</a> systems and also tried to activate the expression upon <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/induction#hormon">chemical stimuli</a>.</p>
<p>In order to allow for control of gene regulation in a time- and space-dependent manner, we designed various control elements. We engineered and tested <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/induction#light">light induction</a> systems and also tried to activate the expression upon <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/induction#hormon">chemical stimuli</a>.</p>
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<p>As dCas9 is a relatively large protein consisting of 1367 amino acids with a molecular weight of 160 kDa, it influences expression rates and toxicity. We designed several <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/method#truncation">truncated versions</a> of dCas9 with the aim to minimize size and increase the expression rate with retained binding capacity.</p>
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<p>As dCas9 is a relatively large protein consisting of 1367 amino acids with a molecular weight of 160 kDa, it influences expression rates and toxicity. We designed several <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/method#truncation">truncated versions</a> of dCas9 with the aim to minimize its size and increase the expression rate while retaining the RNA/DNA binding ability<!--capacity-->.</p>
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<p>Last but not least we established a novel ELISA-based method to assess the binding efficiency of the Cas9 proteins: The uniCAS Binding Assay “<a id="link" href="https://2013.igem.org/Team:Freiburg/Project/method#elisa">uniBAss</a>”. uniBAss was shown to be an innovative tool for the characterization of the binding capacity of our dCas9 fusion proteins and the truncated dCas9 versions. The method offers the possibility for high throughput capabilities and technological expansion to DNA-binding proteins other than Cas9 such as TAL effectors (TALEs) and zinc fingers (ZFNs). </p><br><br>
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<p>Last but not least we established a novel ELISA-based method to assess the binding efficiency of the dCas9 proteins: The uniCAS Binding Assay “<a id="link" href="https://2013.igem.org/Team:Freiburg/Project/method#elisa">uniBAss</a>”. uniBAss was shown to be an innovative tool for the characterization of the binding capacity of our dCas9 fusion proteins and the truncated dCas9 versions. The method offers the possibility for high throughput implementation and technological expansion to DNA-binding proteins other than dCas9 such as TAL effectors (TALEs) and zinc fingers (ZFNs). </p><br><br>
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<p>Hidden as an uncharacterized <i>E. coli</i> locus for more than 15 years <span id="refer"> <a href="#(1)"> [1]</a> </span> , Barrangou et al. identified the CRISPR (<b>C</b>lusterd <b>R</b>egularly <b>I</b>nterspaced <b>S</b>hort <b>P</b>alindromic <b>R</b>epeats) array as previously unknown adaptive prokaryotic immune system <span id="refer"> <a href="#(2)"> [2]</a> </span> . Almost half of all prokaryots make use of this defence mechanism. Thereby, invasive DNA can specifically and efficiently be cleaved. It helps controlling the unselective uptake through natural transformation, phage DNA transduction and horizontal gene transfer by conjugation <span id="refer"> <a href="#(3)"> [3]</a> </span>. This unique feature results from the interplay of non-coding RNAs and CRISPR associated proteins (Cas) which are able to cleave invasive DNA. A wide range of different CRISPR subtypes is known <span id="refer"> <a href="#(4)"> [4]</a> </span>, <span id="refer"> <a href="#(5)"> [5]</a> </span>. We used CRISPR type II b of <i>S. pyogenes</i>. </p>
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<p>The recognition and degradation of invasive DNA by CRISPR/Cas occurs in three steps (see Fig. 2):
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<p>Hidden as an uncharacterized <i>E. coli</i> locus for more than 15 years <span id="refer"> <a href="#(1)"> [1]</a></span>, Barrangou et al. identified the CRISPR (<b>C</b>lusterd <b>R</b>egularly <b>I</b>nterspaced <b>S</b>hort <b>P</b>alindromic <b>R</b>epeats) array as a previously unknown adaptive prokaryotic immune system <span id="refer"> <a href="#(2)"> [2]</a></span>. Almost half of all prokaryotes make use of this defense mechanism against unselective uptake through natural transformation, phage DNA transduction or horizontal gene transfer by conjugation. Invasive DNA or even RNA <!--<span id="refer"> <a href="#()">[]</a></span>--> can be specifically recognized and efficiently cleaved <span id="refer"> <a href="#(3)"> [3]</a></span>. This unique feature results from the interaction of non-coding RNAs and CRISPR associated (Cas) proteins. <span id="refer"> <a href="#(4)">[4]</a></span>, <span id="refer"> <a href="#(5)">[5]</a></span>. From a wide range of known CRISPR subtypes we used CRISPR type II b of <i>S. pyogenes</i>.
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<!--Hier muss mehr him!--></p>
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<p>The recognition and degradation of invasive DNA by CRISPR/Cas type II occurs in three steps (see Fig. 2):
<ol>
<ol>
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<li><b>Acquisition</b>: Invasive DNA is recognized via a protospacer adjacent motif (PAM) – the sequence NGG. This sequence is then integrated into the host CRISPR array and is termed spacer. Spacer sequences transcribe for CRISPR RNAs(crRNAs) which help to sequence specifically cleave invasive DNA. Spacer sequences are located between short palindromic repeats. These palindromic repeats are needed for the functionality of the crRNAs.</li>
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<li><b>Acquisition</b>: Invasive DNA is recognized via a protospacer adjacent motif (PAM) – the sequence NGG. A short sequence downstream of the PAM sequence is then integrated into the host CRISPR array and is termed spacer. Spacer sequences transcribe for CRISPR RNAs(crRNAs) which help to cleave sequence-specific invasive DNA. These sequences are located between short palindromic repeats, which are neccessary for the functionality of the crRNAs.</li>
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<li><b>Expression/Transcription</b>: The Cas9 endonuclease is expressed. CRISPR array is then transcribed and processed  by RNAse III into crRNAs. These contain the complementary spacer sequence and the direct repeat sequence. The crRNA guides the Cas9 protein specifically to invasive DNA sequences. Furthermore trans-activating crRNAs (tracrRNA) are transcribed and bind to the direct repeat part of the crRNA <span id="refer"> <a href="#(6)"> [6]</a></span>. The tracrRNA is necessary for the formation of a Cas9-RNA complex. </li>
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<li><b>Expression/Transcription</b>: The Cas9 endonuclease is expressed and spacer sequences are transcribed to crRNAs. crRNAs contain the complementary spacer sequence and the direct repeat sequence. The crRNA is processed by RNAse III and functions as a guide for the Cas9 protein to specifically trace invasive DNA sequence. Furthermore trans-activating crRNAs (tracrRNA) are transcribed and bind to the direct repeat part of the crRNA <span id="refer"> <a href="#(6)"> [6]</a> </span>. This second type of RNA is required to build a structure that can be bound by the Cas protein. </li>
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<li><b>Interference</b>: Repeatedly invading DNA, which has been integrated into the CRISPR locus, is detected by the RNA-protein complex and cleaved by Cas9.</li>
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<li><b>Interference</b>: Appropriate invasive DNA is detected by the RNA-protein-complex. Now the endonuclease Cas9 disposes the invasive DNA by cleaving.</li>
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<td> <b>Figure 2: The CRISPR/Cas9 system</b><br>
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<td> <b>Figure 3: The CRISPR/Cas9 system</b><br>
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First step is the aquisition of invasive DNA sequences into the CRISPR array. Next step is the transcription of crRNA and tracrRNA. Together they form an RNA hybrid that interacts with Cas9 and enables the protein to bind and cleave invasive DNA.   
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First step is the aquisition of invasive DNA sequences into the CRISPR array. Next step is the transcription of crRNA and tracrRNA. Together they form an RNA hybrid that interacts with Cas9 and enables the protein to bind and cleave invasive DNA in the interference step.   
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<p><b>In short</b>: This CRISPR/Cas9 system is based on the interplay of three acting components: tracrRNA, crRNA and Cas9 protein. Studies showed that RNAse III is sufficiently available in host cells <span id="refer"> <a href="#(7)"> [7]</a> </span>. crRNA helps to detect complementary invasive DNA, tracrRNA enables the interaction of crRNA and Cas9. RNA-Cas9 complex binds appropriate DNA and Cas9 cleaves the DNA sequcence specifically.</p>
 
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<p>Unlike ZFNs, TALEs or Meganucleases, Cas9 proteins perform DNA sequence specific binding by utilizing a unique RNA scaffold composed of crRNA and tracrRNA. With about 20 nucleotides corresponding to the target element and the Cas9's helicase activity, 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, like our <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/crrna#multiple_targeting">RNAimer</a>. 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 <span id="refer"> <a href="#(6)"> [6]</a> </span>.</p>
 
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<p>We believe that in the nearby future CRISPR/Cas9 systems can commonly be used to target multiple DNA sequences. Co-transfecting standardized crRNA-array- and tracrRNA-encoding plasmids with a Cas9-encoding plasmid results in a powerful device for multiplex genome engineering <span id="refer"> <a href="#(7)"> [7]</a> </span>, <span id="refer"> <a href="#(8)"> [8]</a> </span>. 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 <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/crrna#design_tool">crRNA Designing Tool</a>. 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>
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<!-- gehört das noch zu in short? -->
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<!--
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<p>Unlike ZFNs, TALEs or Meganucleases, Cas9 proteins perform DNA sequence specific binding by utilizing a unique RNA scaffold composed of crRNA and tracrRNA. With about 30 nucleotides corresponding to the target element and the Cas9's helicase activity, 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, like our <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/crrna#multiple_targeting">RNAimer</a>. So far, the only constraint for recognition between crRNA and target DNA is a protospacer adjacent motif - PAM -, located directly 3‘ of the protospacer sequence 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 <span id="refer"> <a href="#(6)"> [6]</a></span>.</p>
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<div id="outlook">
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Outlook
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<p> Our toolkit opens up the possibility to deal with a wide range of yet unadressed scientific questions in the near future, including the field of systems biology and complex metabolic engineering 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 <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/crrna#design_tool">crRNA Design Tool</a>. Financial, logistic and human ressources stay at a minimum using the uniCas toolkit for gene regulation. 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>.
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<p> The system could possibly be used for tissue engineering and stem cell reprogramming. Simultaneous regulation of the expression of different pathway enzymes may enhance industrial purposes - e.g. for the production of certain chemical compounds such as amino acids, biofuels or therapeutic drugs in eukaryotic cells. Therefore, stably transfected cells would be of interest.</p>
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<p> Moreover, not only metabolic pathways could be targeted. Developmental Biology studies the embryonic patterning and organ formation. By transient dCas9-G9a interaction with different stem cell factor expressions, possibilities for better understanding and modeling of organism development arise. Induced pluripotent stem cells, for example, are yet made responsible for causing mutagenesis or immunogenicity <span id="refer"> <a href="#(9)"> [9]</a></span>. Regenerative medicine still deals with basic understanding of reprogramming most cell types and may therefore gain benefits from efficient simultaneously acting dCas9-regulators.</p>
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<p> Complex networks, as for instance can be found in cancer cell differentiation, migration and metabolism may be targeted. Frequently, transcriptional master regulators of house-keeping functions and signaling pathways, such as p53, show down- or upregulation in certain tumors <span id="refer"> <a href="#(10)">[10]</a></span>. From a speculative point of view, one could therefore suggest to perform sophisticated dCas9-fusion protein high-throughput transcriptome analysis of cancer-related promoters. Targeting should then first be obtained in cancer cell lines with a multiple crRNA plasmid library. Consequently, promising combinations of crRNAs might then be investigated more precisely and maybe, on a long-term perspective, lead to future therapeutic relevance.</p>
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<div id="(7)">(7) Cong, L., <i>et al.</i> (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, <i>819-823</i>.<br></div>
<div id="(7)">(7) Cong, L., <i>et al.</i> (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, <i>819-823</i>.<br></div>
<div id="(8)">(8) Mali, P., <i>et al.</i> (2013). RNA-guided human genome engineering via Cas9. Science 339, <i>823-826</i>.<br></div>
<div id="(8)">(8) Mali, P., <i>et al.</i> (2013). RNA-guided human genome engineering via Cas9. Science 339, <i>823-826</i>.<br></div>
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<div id="(9)">(9) Yamanaka, S., <i>et al.</i>  (2012). Induced Pluripotent Stem Cells: Past, Present and Future. Cell Stem Cell 10,
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<i>678-684</i>.<br></div>
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<div id="(10)">(10) Kaneshiro, K., <i>et al.</i> (2007). An integrated map of p53-binding sites and histone modification in the human ENCONDE regions. Genomics,
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<i>177-188</i>.<br></div>
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Latest revision as of 03:22, 29 October 2013


Introduction

Abstract

Our team developed a universal toolkit, termed uniCAS, that enables customizable gene regulation in mammalian cells. Therefore, we engineered the recently understood and highly promising CRISPR/Cas9 system. The regulation is based on the RNA-guided Cas9 protein, which allows specific targeting of DNA sequences. Our toolkit comprises not only a standardized Cas9 protein, but also different effector domains for efficient gene activation or repression. We further engineered a modular RNA plasmid for easy implementation of RNA guide sequences. As an additional feature, we established an innovative screening method for assessing the functionality of our uniCAS fusion proteins. Single genes and even whole genetic networks can be modified using our uniCAS toolkit. We think that our toolbox of standardized parts of the CRISPR/Cas9 system offers broad application in research fields such as tissue engineering, stem cell reprogramming and fundamental research.

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Introduction

Introduction to our project

The enormous amount of gene regulation, signal transduction and metabolic pathways gives us a slight idea about what complexity of life really means. This astonishing complexity is one of the most crucial issues to understand, if we want to understand life itself.

Many approaches have been investigated and given us insights into these biological pathways, resulting in more and more elaborate tools. Solutions for the treatment of systemic diseases like cancer or the fine-tuning of cellular processes remain challenging. There is a need for a powerful tool, which enables powerful regulation of whole genetic networks and yet is simple to use. In the last years there have been several approaches which were able to alter gene expression, but they have the flaw that they are only targeting one gene at a time and do not allow flexible modification of entire genomes.

In the last 8 months, our team has been planning, developing and building up a universal toolkit, which enables the regulation of complex networks. A toolkit that allows the simple and flexible regulation of multiple genes by only one protein.

Figure 1: Our uniCAS toolkit
The uniCAS toolkit provides 3 different effectors, 2 methods & 1 effector control! By using our toolkit it is possible to efficiently activate or repress gene expression. We also provide devices for controlling our effectors by light. For multiple targeting different DNA sites can be addressed simultaneously by using our RNAimer plasmid.
Try working with uniCAS and receive custom-tailored instructions for your desired gene regulation experiment by using our Manual Tool.


The great potential harbored by the CRISPR/Cas9 system provided a great basis for us to modify it in a way that allows multiple, sequence specific gene regulation in mammalian cells.

The CRISPR/Cas9 system in short:
This CRISPR/Cas9 system is based on the interaction of three acting components: tracrRNA, crRNA and Cas9 protein. The crRNA detects complementary invasive DNA, tracrRNA enables the interaction of crRNA and Cas9. The RNA-Cas9 complex binds respective DNA and Cas9 cleaves the DNA sequcence specifically.


We used a human codon optimized Cas9, mutated the remaining nickase function and standardized the 160 kDa protein. The result: The DNA binding protein dCas9 which can be guided specifically to any DNA target site due to complexation with crRNA (CRISPR RNA) and tracrRNA (transactivating crRNA). By fusing different effector domains to dCas9 we built fusion proteins for gene regulation in mammalian cells.

Figure 2: Engineering of the CRISPR/Cas9 system
The nuclease function of Cas9 has been mutated. The dCas9 is still able to bind the guiding RNAs and therewith can be directed towards every requested sequence. By fusing effectors to the dCas9, gene regulation of specitic loci can be engineered.

For gene regulation we used the trans-activator VP16, the repressor-domain KRAB and the histone methyltransferase G9a. These effectors were able to efficiently change the expression rate of a reporter protein SEAP (secreted alcaline phosphatase) and the endogenous signal protein VEGF (vascular endothelial growth factor).

In order to allow for control of gene regulation in a time- and space-dependent manner, we designed various control elements. We engineered and tested light induction systems and also tried to activate the expression upon chemical stimuli.

As dCas9 is a relatively large protein consisting of 1367 amino acids with a molecular weight of 160 kDa, it influences expression rates and toxicity. We designed several truncated versions of dCas9 with the aim to minimize its size and increase the expression rate while retaining the RNA/DNA binding ability.

Last but not least we established a novel ELISA-based method to assess the binding efficiency of the dCas9 proteins: The uniCAS Binding Assay “uniBAss”. uniBAss was shown to be an innovative tool for the characterization of the binding capacity of our dCas9 fusion proteins and the truncated dCas9 versions. The method offers the possibility for high throughput implementation and technological expansion to DNA-binding proteins other than dCas9 such as TAL effectors (TALEs) and zinc fingers (ZFNs).



Background: The CRISPR/Cas9 system

Hidden as an uncharacterized E. coli locus for more than 15 years [1], Barrangou et al. identified the CRISPR (Clusterd Regularly Interspaced Short Palindromic Repeats) array as a previously unknown adaptive prokaryotic immune system [2]. Almost half of all prokaryotes make use of this defense mechanism against unselective uptake through natural transformation, phage DNA transduction or horizontal gene transfer by conjugation. Invasive DNA or even RNA can be specifically recognized and efficiently cleaved [3]. This unique feature results from the interaction of non-coding RNAs and CRISPR associated (Cas) proteins. [4], [5]. From a wide range of known CRISPR subtypes we used CRISPR type II b of S. pyogenes.

The recognition and degradation of invasive DNA by CRISPR/Cas type II occurs in three steps (see Fig. 2):

  1. Acquisition: Invasive DNA is recognized via a protospacer adjacent motif (PAM) – the sequence NGG. A short sequence downstream of the PAM sequence is then integrated into the host CRISPR array and is termed spacer. Spacer sequences transcribe for CRISPR RNAs(crRNAs) which help to cleave sequence-specific invasive DNA. These sequences are located between short palindromic repeats, which are neccessary for the functionality of the crRNAs.
  2. Expression/Transcription: The Cas9 endonuclease is expressed. CRISPR array is then transcribed and processed by RNAse III into crRNAs. These contain the complementary spacer sequence and the direct repeat sequence. The crRNA guides the Cas9 protein specifically to invasive DNA sequences. Furthermore trans-activating crRNAs (tracrRNA) are transcribed and bind to the direct repeat part of the crRNA [6]. The tracrRNA is necessary for the formation of a Cas9-RNA complex.
  3. Interference: Repeatedly invading DNA, which has been integrated into the CRISPR locus, is detected by the RNA-protein complex and cleaved by Cas9.
Figure 3: The CRISPR/Cas9 system
First step is the aquisition of invasive DNA sequences into the CRISPR array. Next step is the transcription of crRNA and tracrRNA. Together they form an RNA hybrid that interacts with Cas9 and enables the protein to bind and cleave invasive DNA in the interference step.

Outlook

Our toolkit opens up the possibility to deal with a wide range of yet unadressed scientific questions in the near future, including the field of systems biology and complex metabolic engineering 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 Design Tool. Financial, logistic and human ressources stay at a minimum using the uniCas toolkit for gene regulation. 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.

The system could possibly be used for tissue engineering and stem cell reprogramming. Simultaneous regulation of the expression of different pathway enzymes may enhance industrial purposes - e.g. for the production of certain chemical compounds such as amino acids, biofuels or therapeutic drugs in eukaryotic cells. Therefore, stably transfected cells would be of interest.

Moreover, not only metabolic pathways could be targeted. Developmental Biology studies the embryonic patterning and organ formation. By transient dCas9-G9a interaction with different stem cell factor expressions, possibilities for better understanding and modeling of organism development arise. Induced pluripotent stem cells, for example, are yet made responsible for causing mutagenesis or immunogenicity [9]. Regenerative medicine still deals with basic understanding of reprogramming most cell types and may therefore gain benefits from efficient simultaneously acting dCas9-regulators.

Complex networks, as for instance can be found in cancer cell differentiation, migration and metabolism may be targeted. Frequently, transcriptional master regulators of house-keeping functions and signaling pathways, such as p53, show down- or upregulation in certain tumors [10]. From a speculative point of view, one could therefore suggest to perform sophisticated dCas9-fusion protein high-throughput transcriptome analysis of cancer-related promoters. Targeting should then first be obtained in cancer cell lines with a multiple crRNA plasmid library. Consequently, promising combinations of crRNAs might then be investigated more precisely and maybe, on a long-term perspective, lead to future therapeutic relevance.


References

(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) Jansen, R., et al. (2002). Identification of genes that are associated with DNA repeats in prokaryotes. Molecular Microbiology 43, 1565-1575.
(5) Makarova, K., et al. (2011). Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9, 467-477.
(6) Jinek, M., et al. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821.
(7) Cong, L., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823.
(8) Mali, P., et al. (2013). RNA-guided human genome engineering via Cas9. Science 339, 823-826.
(9) Yamanaka, S., et al. (2012). Induced Pluripotent Stem Cells: Past, Present and Future. Cell Stem Cell 10, 678-684.
(10) Kaneshiro, K., et al. (2007). An integrated map of p53-binding sites and histone modification in the human ENCONDE regions. Genomics, 177-188.