Team:Kent/HP/Silver

• Project
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• Team
  • Meet the Team
  • Contribution
  • Attribution
• Human Practices
  • Integrated HP
  • Public Engagement
  • Interlab
  • Collaboration

Integrated HP

Our team has strived to make human practices the solid foundation of our project right from the start. We wanted to ensure we fully understood the implications of any project we chose to pursue while addressing current global problems.
We held a brainstorming session where we bounced ideas off of one another for potential project ideas and had narrowed it down to a top five. We decided we needed to consult with our immediate community to scope out what issues they deemed important that needed immediate addressing. Through different outreach opportunities, we were able to comprehend the controversy surrounding GMOs and genetic engineering in general. This caused us to critically analyze our project ideas before deciding upon ‘LuCAS’: a novel way of mRNA localization using CRISPR - dCas13a.

Why CRISPR-dCas13a (C2C2)?

We were introduced to CRISPR-Cas9 as a tool for targeting cellular mRNA translation through Dr. Peter Ellis, a lecturer in Molecular Biology and Reproduction. However, through research, we discovered that Cas9 is a DNA endonuclease, meaning it recognizes DNA targets and cuts them. It would need to be ‘tricked’ by molecular means. C2C2, however, or Cas13a is an RNA endonuclease, meaning it cuts the RNA rather than the DNA. There are two key differences when comparing Cas13 to CAS9: Firstly, Cas13 recognizes RNA rather than DNA. Secondly, once the target is recognized, it acts promiscuously and starts to ‘chew up’ all of the RNA around it, not only the intended target sequence.

We wanted to utilize this knowledge and build upon the CRISPR-Cas9 and produce a tool that would just track the mRNA, not cut it. We did not want to overcomplicate our methodology and deemed tagging the mRNA with GFP would be sufficient as we would be able to essentially track the mRNA movement to ensure its localization without damaging its integrity through cutting it.

Surveys and Consultations

Surveys

• We wanted the opportunity to receive insight from the public immediately, even before deciding on a project idea. We wanted to ensure that any topic we would be putting our focus into would heed positive results, not only from the scientific community but from the general public as well.
• We set out to design several surveys, questioning the community at our University and locally on their knowledge of GMOs and whether our project abstract was an idea they agreed would be beneficial once produced.
• The general consensus from the students at the University was that they agreed genetic modification would be beneficial when used appropriately, however strict regulations would need to be in place when dealing with GMO applications, a small percentage even saying they would go the extra mile to ensure they did not deal with GMOs in their day to day lives.
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• With this in mind, we introduced our project abstract and explained the positive outcomes we saw it producing and its potential as a diagnostic tool. The majority of the feedback we received was positive and allowed us to confidently advance in our laboratory work with a goal in mind: produce a tool with a promising future in diagnositcs.
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Professor Michelle Garrett

When conceptualizing our project, we realized we would need to transfect our cell lines into mammalian cells if we wanted to have any use of it as a potential diagnostic tool, as it would be relatively useless in its bacterial cell host. Professor Garrett was able to guide us in the direction of the Smales Group at our University, providing us with the resources needed to make the transfections possible.

Ian Brown, Microscopy Suite Facility Manager

As CRISPR itself has only recently been discovered as an mRNA tracking tool, most of the project entailed repeated trial and error. We realized before we even thought of imaging, we needed to ensure we had the appropriate controls for comparative and evaluative reasons. We were able to generate a repeatable layout for future imaging purposes that Ian Brown was able to confirm would be adequate in imaging our cells accurately

Dr. Emma Mead

Through research, we were able to conclude the best possible cells to transfect our constructs into would have to be HEK293 cells. However, without experience in such a field, we were guided to Dr. Mead, who proved to be invaluable throughout the entirety of our project. She confirmed that it would indeed have to be the HEK293 cells and provided us with samples to work with, as well as guiding us through the passaging and transfection itself.

Dr. Rosalyn Masterton

We were able to conclude that we would need a vector to clone our construct before we could image it and decided upon pcDNA 3.1 (+) mammalian vector through the help of Dr. Masterton with her expertise in molecular biology. She also advised us who to speak to for the use of mammalian cell lines.