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| − | <h1>gRNA Testing System</h1>
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| | + | <section class="page-header page-header-lg parallax parallax-3" style="background-image:url('https://static.igem.org/mediawiki/2016/2/23/T--Valencia_UPV--notebookTitleBack.png');"> |
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| | + | <h1>Split Cas9</h1> |
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| − | <button class="fa fa-bars"></button>
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| − | <h4>Index</h4>
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| − | </div>
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| − | <ul class="list-group list-group-bordered list-group-noicon uppercase">
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| − | <li class="list-group-item"><a href="#"><span class="size-11 text-muted pull-right"></span> First Stuff</a></li>
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| − | <!-- POST ITEM -->
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| − | <!-- IMAGE -->
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| − | <figure class="margin-bottom-20">
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| − | <img class="img-responsive" src="assets/images/demo/content_slider/10-min.jpg" alt="">
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| − | </figure>
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| − | <h3>gRNA Testing System</h3>
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| − | <p>
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| − | <h3>Abstract</h3>
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| − | </p>
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| − | <p>We have designed a modular gRNA testing system in order to check if the gRNA provided by the
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| − | Data Processing Software works as expected on the plant variety to improve. This system works
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| − | as a <b>genetic switch</b> that remains OFF if the gRNA does not work, and turns ON when
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| − | the Cas9 does a double stranded break in the target. The device performance is based on the
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| − | fact that the <b>luciferase reporter gene</b> is out of its reading frame. When CRISPR/Cas9
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| − | system works as expected, meaning that the <b>gRNA is well designed</b>, it will introduce
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| − | indels in our device and the luciferase will be placed on its correct reading frame. Therefore,
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| − | we will be able to detect bioluminescence with a luciferase assay. </p>
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| − | <br>
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| − |
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| − | <p><b><u>gRNA Testing System</b></u>
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| − | </p>
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| − | <br>
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| − | <p>gRNA is a key element in genome editing with CRISPR/Cas9 system, since it leads endonuclease
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| − | Cas9 where the modification must be produced. That means gRNA must work properly in the plant
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| − | variety that we want to improve. </p>
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| − | <br>
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| − |
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| − |
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| − | <p>The plant breeder could use the gRNA provided by our software directly in his plant. However,
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| − | lots of plant species take a long time to grow. For example, orange trees need at least 1-2
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| − | years to exceed the first growth stage, and 3-4 years to produce the first fruits. If the
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| − | gRNA didn’t work properly, the plant breeder would had lost a valuable time waiting
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| − | to see a phenotypic improvement on his plant. It would take a long time just to know if the
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| − | gRNA has worked or not. Due to that, it’s necessary to previously check the gRNA proper
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| − | functioning.</p>
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| − | <br>
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| − |
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| − |
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| − | <p><b>Why would not our gRNA work?</b></p>
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| − | <br>
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| − | <p>Our open-source database includes genes of interest, which can be selected by plant breeders
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| − | according to their needs. It also directly provides the optimal gRNA which, afterwards, must
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| − | be ordered to synthesize. The gRNA is obtained from gene consensus sequences acquired from
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| − | big databases such as NCBI or Sol Genomic. However, the consensus sequence may match or not
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| − | with the specific variety to improve. Insertions and deletions - Indels - and Single Nucleotide
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| − | Polymorphisms - SNPs - occur spontaneously and randomly in the genome of different plant
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| − | varieties. That means the gRNA obtained by our data processing software could not work optimally
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| − | on the desired variety. This makes even more necessary to test the gRNA before using it in
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| − | the specific variety. Furthermore, even if the gRNA matches perfectly with the target sequence,
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| − | mutagenesis may not occur - or be less efficient - due to gRNA secondary structure problems.</p>
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| − | <br>
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| − |
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| − |
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| − | <p><b><u>gRNA testing methods</b></u>
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| − | </p>
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| − | <br>
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| − | <p>The current methods used to test the efficiency of CRISPR/Cas9 -and therefore that can be used
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| − | to test the gRNA- are digestion with restriction enzymes or digestion with T7 endonuclease
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| − | and subsequent sequencing. However, these methods are not suitable if we want to make accessible
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| − | and easy the testing of the gRNA. </p>
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| − | <br>
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| − |
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| − |
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| − |
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| − |
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| − | <ul>
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| − | <li><b>Digestion with restriction enzymes</b>: when choosing the target within the gene, it is
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| − | mandatory to choose a target including a restriction site where the Cas9 will make the
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| − | DSB. Therefore, in order to check if the Cas9 produced the mutation, a digestion is performed
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| − | with the restriction enzyme, and an electrophoresis gel is carried out. If the Cas9 cuts,
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| − | the restriction site will be lost and the enzyme will not cut. Therefore, in the electrophoresis
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| − | gel, a band with higher molecular weight should be observed. If mutation did not occur,
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| − | two bands should be observed, since the enzyme can recognize the site and cut. </li>
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| − |
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| − | <ul>
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| − | <li><b>Problem</b>: the range of possible targets is reduced, because they must contain a
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| − | restriction site exactly in the place where the Cas9 cuts. Additionally, when you
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| − | find a target with a restriction site you might not have the needed enzyme. Buying
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| − | it may imply a high cost, not affordable for everyone. </li>
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| − | <br>
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| − | </ul>
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| − | </ul>
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| − |
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| − | <ul>
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| − | <li><b>Digestion with T7 endonuclease</b>: this strategy is similar to the restriction enzymes
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| − | one, yet it uses the T7 endonuclease. This endonuclease cuts where it finds heterodimers.
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| − | When Cas9 cuts, due to the non-homologous end joining DNA repair mechanism - NHEJ -,
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| − | plant cells introduce indels. After a PCR amplification of the region, heterodimers can
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| − | be obtained from a denaturation and reannealing step. When they are annealed, they might
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| − | bind with a strand which is not exactly complementary, producing heterodimers that T7
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| − | can cut. Therefore, in an electrophoresis gel, a high molecular weight band will be observed
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| − | if there is not a cut, while, two shorter bands will appear if T7 endonuclease cuts and
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| − | so, Cas9 is well working.</li>
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| − |
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| − | <ul>
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| − | <li><b>Problem</b>: the T7 endonuclease is outrageously expensive. Buying it may imply even
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| − | a higher cost, not affordable for everyone. </li>
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| − |
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| − | </ul>
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| − | </ul>
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| − |
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| − |
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| − | <br>
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| − | <p>In order to provide an efficient solution to the drawbacks explained above, we have engineered
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| − | a gRNA Testing System. In this strategy, we use <i>Nicotiana benthamiana</i> due to its fast
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| − | growth and all the benefits provided by a model plant. Our methodology is based in the GoldenBraid
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| − | Assembly System, that allows us to get a modular and standard system, two of the mainstays
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| − | of Synthetic Biology field. </p>
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| − | <br>
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| − |
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| − |
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| − | <p><b><u>Our device</b></u>
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| − | </p>
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| − | <br>
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| − | <p>The fragment of genome that we are going to target in our plant is inserted in the Testing System
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| − | following the concept of modularity. Based on <i><i>Agrobacterium</i> tumefaciens</i> infection
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| − | and using a reporter gene in our device, we will be able to know how efficient the provided/designed
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| − | gRNA is. </p>
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| − | <br>
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| − | <p>First of all, plant breeders have to carry out a genomic DNA extraction of the plant they want
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| − | to modify. Next, they need to amplify the region they are going to target. The targets needed
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| − | to this amplification are provided by our Data Processing Software. The amplified target
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| − | sequence is introduced in <i>N. benthamiana</i> as part of the device with the luciferase
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| − | reporter. </p>
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| − | <br>
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| − | <img class="img-responsive" style="float:left;width:55%;" src="https://static.igem.org/mediawiki/2016/4/49/Gts_target_1_upv.png">
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| − |
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| − |
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| − | <p>This device is introduced in the plant along with the corresponding gRNA and Cas9 construction.
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| − | If the gRNA works on the desired variety, we will be able to detect it easily with a luciferase
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| − | assay.</p>
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| − | <br>
| |
| − | <img class="img-responsive" style="float:left;width:55%;" src="https://static.igem.org/mediawiki/2016/a/a9/Gts_target_2_upv.png">
| |
| − | <p>Our system is designed to allow the detection of luminescence. Thus, a luciferase assay is used
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| − | to study gene expression rates, since it is fast and the analysis of each sample only requires
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| − | a few minutes. Moreover, it is extremely sensitive and the results are very accurate, allowing
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| − | us to obtain quantitative results. Originally the system is OFF since luciferase genetic
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| − | sequence is not in the correct frame. <b>When Cas9 cuts, indels appear, system turns ON, so luciferase gene is in the correct frame and it will be correctly translated.</b> In that case, the plant breeder could check the genome editing in a simple way. Cells are
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| − | assayed for the presence of the reporter by directly measuring the enzymatic activity of
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| − | the reporter protein on luciferin substrate. </p>
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| − | <br>
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| − | <br>
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| − |
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| − |
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| − | <p><b><u>Parts of the device</b></u>
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| − | </p>
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| − | <br>
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| − | <p>P35s : 5’ region : TARGET : Linker (+2) : LUC : Tnos</p>
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| − | <br>
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| − | <br>
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| − |
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| − | <ul>
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| − | <li><b>P35s</b>: It is a strong constitutive promoter derived from cauliflower mosaic virus (CaMV).
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| − | It is widely used in plants to improve the level of the expression of foreign genes effectively
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| − | in all tissues. </li>
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| − | <br>
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| − | </ul>
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| − |
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| − | <ul>
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| − | <li><b>5’ region</b>: 5’ region from the <i>N. benthamiana</i> polyubiquitin sequence
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| − | (Accession number: Nbv5.1tr6241949) is used due to its high expression rate in every
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| − | plant tissue. It can help our gene to express itself and ensures that the construction
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| − | is expressed. It contains BsmbI and BsaI recognition sites with AATG in 3’ as overhang
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| − | that allows us to ligate with the amplified target.</li>
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| − | <br>
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| − | </ul>
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| − |
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| − | <ul>
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| − | <li><b>Target</b>: Plant breeders will obtain the selected gene from their original plant by
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| − | PCR amplification with the primers provided by our Data Processing Software. These primers
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| − | are designed in order to obtain amplicons with the overhangs needed to insert the target
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| − | in our device, corresponding to GoldenBraid grammar (prefix: AATG, suffix: TTCG). It’s
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| − | mandatory to make sure that this region doesn’t contain any stop codon in frame
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| − | +1, but neither in frame +2, so when an indel happens the reading frame will not be disrupted.
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| − | The software finds the optimal target with its corresponding gRNA.</li>
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| − | <br>
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| − | </ul>
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| − |
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| − | <ul>
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| − | <li><b>Linker SAGTI (Ser-Ala-Gly-Thr-Ile)</b>: it is a flexible peptide linker between the target
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| − | and the luciferase, so it allows the luciferase to acquire the correct structure, avoiding
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| − | interaction with the target. Thus, luciferase assay will be carried out in a successfully
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| − | way. As it can be seen in the figure 1, the first part of the device is in ORF +1 whereas
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| − | the luciferase is in ORF +2. The device is designed so that there is an extra nucleotide
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| − | before the linker sequence to change the reading frame. If this nucleotide were after
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| − | the linker sequence, when Cas9 cut, the reading frame of the linker would change, and
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| − | the amino acids translated would not be the correct ones. Figure 1 shows how it will
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| − | be translated after the indels.</li>
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| − | <br>
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| − | </ul>
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| − | <img class="img-responsive" src="https://static.igem.org/mediawiki/2016/4/47/Gts_target_3_upv.png">
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| − |
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| − |
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| − |
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| − | <p>Figure 1. Device after endonuclease Cas9 cut and indels production. a) The extra nucleotide is
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| − | located before the linker, so when indels change the frame, the linker and the luciferase
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| − | are in the correct ORF. b) The extra nucleotide is located after the linker. Thus linker’s
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| − | frame will be different from luciferase’s one after indel occurs.</p>
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| − | <br>
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| − | <p>Luciferase: It is widely used as a reporter enzyme. At the beginning, luciferase gene is out
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| − | of the reading frame due to the presence of an extra nucleotide. After CRISPR/Cas9 acts indels
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| − | occur. These insertions and deletions of nucleotides produce a frameshift and change luciferase’s
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| − | frame in the right frame +1, so it will be correctly translated. In the experimental design,
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| − | the first methionine has been removed just to prevent the unintended translation of the luciferase.
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| − | Therefore appearance of false positives will be avoided. </p>
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| − | <br>
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| − |
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| − |
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| − | <ul>
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| − | <li><b>Tnos</b>: Nopaline synthase terminator of the nopaline synthase gene of <i><i>Agrobacterium</i> tumefaciens</i>. It is used for gene transfection. </li>
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| − | <br>
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| − | </ul>
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| − |
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| − | <p>The plant breeder will be provided with a pUPD2 vector with P35s:5’region, another pUPD2
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| − | with linker:luciferase, and a pUPD2 ready to insert the target directly obtained from their
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| − | plant variety. Using GoldenBraid assembly, the breeder will insert the target within the
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| − | pUPD2. Afterwards, in a GoldenGate ligation reaction, all the parts of our device can be
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| − | assembled, obtaining the complete gRNA testing system construction. Afterwards <i>A. tumefaciens</i> will be transformed with the obtained plasmid and it will be used to agroinfiltrate <i>N. benthamiana</i> leaves. Four days post infiltration, the breeder will be able to perform the luciferase assay.
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| − | </p>
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| − | <br>
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| − |
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| − |
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| − |
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| − |
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| − | <p><b><u>Bibliography</b></u>
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| − | </p>
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| − | <br>
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| − |
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| − |
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| − | <ul>
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| − | <li>Biolabs, N.(2016).Measuring Targeting Efficiency with the T7 Endonuclease I Assay | NEB.
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| − | (online) Neb.com. Available at: https://www.neb.com/applications/cloning-and-synthetic-biology/genome-editing/measuring-targeting-efficiency-with-the-t7-endonuclease-i-assay
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| − | (Accessed 27 Jul. 2016).</li>
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| − |
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| − | </ul>
| |
| − |
| |
| − | <ul>
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| − | <li>Pauli, S., Rothnie, H., Chen, G., He, X. and Hohn, T. (2004). The Cauliflower Mosaic Virus
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| − | 35S Promoter Extends into the Transcribed Region. Journal of Virology, 78(22), pp.12120-12128.</li>
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| − |
| |
| − | </ul>
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| − |
| |
| − | <ul>
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| − | <li>Guilley, H., Dudley, R., Jonard, G., Balàzs, E. and Richards, K. (1982). Transcription of
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| − | cauliflower mosaic virus DNA: detection of promoter sequences, and characterization of
| |
| − | transcripts. Cell, 30(3), pp.763-773.</li>
| |
| − |
| |
| − | </ul>
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| − |
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| − | <ul>
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| − | <li>Sefapps02.qut.edu.au. (2016). Benthamiana Atlas. (online) Available at: http://sefapps02.qut.edu.au/atlas/tREXXX2new.php?TrID=Nbv5.1tr6241949
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| − | (Accessed 27 Jul. 2016).</li>
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| − |
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| − | </ul>
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| − |
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| − | <ul>
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| − | <li>Chen, X., Zaro, J. and Shen, W. (2013). Fusion protein linkers: Property, design and functionality.
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| − | Advanced Drug Delivery Reviews, 65(10), pp.1357-1369.</li>
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| − |
| |
| − | </ul>
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| − |
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| − | <ul>
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| − | <li>Holden, M., Levine, M., Scholdberg, T., Haynes, R. and Jenkins, G. (2009). The use of 35S
| |
| − | and Tnos expression elements in the measurement of genetically engineered plant materials.
| |
| − | Anal Bioanal Chem, 396(6), pp.2175-2187.</li>
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| − |
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| − |
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| − |
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| − | </p>
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