View source for Screening! of zebrafish DNA repair mutants generated by CRISPR/Cas9 system - Code : KSI0021

<p><span style="font-size:18px">Project :&nbsp;Screening of zebrafish DNA repair mutants generated by CRISPR/Cas9 system</span></p>

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<p><span style="font-size:18px">The reason why to do this project ?&nbsp;</span></p>

<p>- To know the which is the mutant zebrafish and the mutant information &nbsp;for further study.</p>

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<p><span style="font-size:18px">The necessity of DNA Replication / Recombination / Replication &nbsp;(3R) mutants on zebrafish</span><br />
1.Function of DNA 3R processes have been limitedly studied in living organisms.<br />
2.Due to maternal RNA effect with faster development pace, zebrafish mutant embryos can bypass the lethal phenotype compared to mouse model system.<br />
3.Diverse genetic approaches are available in zebrafish system : CRISPR/Cas9.</p>

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<p><span style="font-size:20px">Overview of mutant screening process</span></p>

<p><img alt="" src="/ckfinder/userfiles/images/ssss.PNG" style="height:559px; width:814px" /></p>

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<p>To know or to make the CRISPR gRNA&nbsp;target site &gt; Using BLAT</p>

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<p><img alt="" src="/ckfinder/userfiles/images/blat1.PNG" style="height:768px; width:1366px" /><img alt="" src="/ckfinder/userfiles/images/ww.PNG" style="height:768px; width:1366px" /><img alt="" src="/ckfinder/userfiles/images/blat3.PNG" style="height:768px; width:1366px" /></p>

<p>If we know the Forward primer , Reverse primer and Target site sequence, we know the which gene and where is the target exon. Like follows.&nbsp;</p>

<p><img alt="" src="/ckfinder/userfiles/images/dddd.PNG" style="height:72px; width:817px" /></p>

<p>After make the zebrafish mutant, we have to screen the zebrafish is WT or Mutant.&nbsp;</p>

<p>After genomic DNA extraction &gt; Genetic Analyzer&nbsp;</p>

<p><img alt="" src="/ckfinder/userfiles/images/ff.PNG" style="height:236px; width:819px" /></p>

<p>This is genetic anaylzer data.&nbsp;</p>

<p>So we can confirm the 5bp del mutation occured.&nbsp;</p>

<p>Next we have to further confirm its sequence.</p>

<p>By comparing with WT sequence, we can confirm which sequence is deleted&nbsp;</p>

<p>And further becuase of that we can identify the framshift. and premature stop codon.</p>

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<p>This is what I have done in laboratory.&nbsp;</p>

<p>I learned PCR, BLAT, Sequencing&nbsp;and so on in genomics class.&nbsp;</p>

<p>I didn&#39;t stop by learning the knowledge of genomics but experimented it with critical and independent thinking.</p>

<p>Like &#39; Is it real? or Is it possible? &#39; or something like that .</p>

<p>I think there are two big implications here.<br />
One is that I have experimented with what I learned in class and it&nbsp;is because it comes from creative and independent thinking.<br />
Second, these mutant screening processes may seem like nothing, but they contribute to the advancement of science.</p>

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