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− | <p>After the workshop conducted at one-north festival, we requested the public to participate in a survey. | + | <p>After the workshop conducted at one-north festival, we requested the public to participate in a survey. This survey was designed to gauge public interest and reservations about the use of Cas9 for therapeutic applications.</p> |
− | <p>After the workshop, we | + | <img src="https://static.igem.org/mediawiki/2017/e/ea/Ntu_ihp_survey1.png" > |
− | <p>We then designed our experiments to test the same 10 sgRNAs in the A549 cell line (that we previously tested in PC9 cells, more details are on our Project tab), while the remaining of the workflow was kept similar. A549 cells are derived from lung carcinomatous tissue and | + | </br> |
+ | </br> | ||
+ | </br> | ||
+ | </br> | ||
+ | <p>An overwhelmingly high number (85%) of participants is receptive to using a fully mature CRISPR/Cas9 technology – that is without any side effects. </p> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/5/5a/Ntu_ihp_survey2.png" > | ||
+ | </br> | ||
+ | </br> | ||
+ | </br> | ||
+ | </br> | ||
+ | <p>When asked to consider whether they would be receptive to using CRISPR/Cas9 technology in the event of personal suffering to lung cancer – even when the issues of the treatment have yet to be fully worked out, more than half stated that they would, while 30% would consider the treatment. </p> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/b/bd/Ntu_ihp_survey3.png" > | ||
+ | </br> | ||
+ | </br> | ||
+ | </br> | ||
+ | </br> | ||
+ | <p>However, with the current state of CRISPR/Cas9 technology, half of the public would not utilize this technology. 40% cited worries about side effects of this technology – such as potential off-target effects. </p> | ||
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+ | <p>Being aware and motivated by the concerns of the majority, we decided to extend our EGFR project as an integration of public opinions into our research so that we can derive a more thoughtful conclusion.</P | ||
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+ | <p>After the workshop, we realized that we may have overlooked the possibility of off-targets. We then brainstormed and came to the idea of targeting our single-guide RNAs (sgRNAs) to wild-type EGFR gene to investigate the possibility of the off-target effect that CRISPR-Cas system could introduce since the desirable CRISPR-Cas systems should only target and perform their cutting work on the mutated allele, not on the wild-type EGFR allele. This is of concern since cancer causing mutations in the EGFR allele includes deletions, and our design of sgRNAs may not be specific enough for disease allele. </p> | ||
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+ | <p>We then designed our experiments to test the same 10 sgRNAs in the A549 cell line (sgRNAs that we previously tested in PC9 cells, more details are on our Project tab), while the remaining of the workflow was kept similar. A549 cells are derived from lung carcinomatous tissue and harbours the wild-type EGFR gene.</p> | ||
<img src="https://static.igem.org/mediawiki/2017/5/54/Goldhuman_NTU.jpg" > | <img src="https://static.igem.org/mediawiki/2017/5/54/Goldhuman_NTU.jpg" > | ||
</br> | </br> | ||
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</br> | </br> | ||
</br> | </br> | ||
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− | <p>The T7E1 results for A549 cells with 24h transfection time were all negative as we expected, suggesting that CRISPR-Cas could not target the wild-type EGFR gene with all the sgRNAs designed. Surprisingly, positive T7E1 results were observed for some sgRNAs when A549 cells were transfected with 48h time, which may indicate that CRISPR-Cas could possibly tolerate the mutation and perform | + | <p>The T7E1 results for A549 cells with 24h transfection time were all negative as we expected, suggesting that CRISPR-Cas could not target the wild-type EGFR gene with all the sgRNAs designed. Surprisingly, positive T7E1 results were observed for some sgRNAs when A549 cells were transfected with 48h time, which may indicate that CRISPR-Cas could possibly tolerate the mutation and perform cleavage. Another parameter which could matter is the transfection time, since the cuts were only observed after 48h transfection time but not in 24h transfection time. This could mean that longer transfection time may affect the accuracy of CRISPR-Cas as well.</p> |
Revision as of 18:28, 1 November 2017