Revision as of 21:00, 1 November 2017

• ABOUT US
  Team Attributions
• BACKGROUND
  Alternative Splicing CRISPR RNA Binding Proteins REST
• PROJECT
  Guides and ASOs mKate-FF4 2 Exon mKate HBG 3 Exon mKate HBG 3 Exon Dual Fluorescence
• LAB WORK
  Parts/Improved Parts Protocols Notebook Future Work
• MODELLING
• HUMAN PRACTICES
  Integrated HP Public Engagement Collaborations InterLab
• AWARDS
  HP Silver HP Gold Education & Public Engagement Model Parts Collection

Human Practices: Gold

Meeting with Experts

Zhang Lab

Dr. Feng Zhang and the Zhang lab were integral in beginning to understand the scope of alternative splicing, and how Cas13a works in cells. We confided with him and other members of his lab about Cas13a (specifically dCas13a), RBPs, and other characteristics related to splicing. These discussions led us to use Cas13a as the primary protein to demonstrate control over splicing.

In addition to gaining advice about Cas13a and alternative splicing, we were introduced to the legal processes involved with biology. We looked to expand our collaboration with the Zhang lab by using a Cas13a protein from another species that the lab was using. Whereas L.shahii Cas13a isn't catalytically active in mammalian cells, the protein the Zhang lab worked with, L. wadei, is catalytically active. Although we weren't concerned with the catalytic activity (i.e. the ability for the protein to cut mRNA), it was believed that the L. wadei would do a better job at controlling exon exclusion in our HEK cells. Unfortunately, the research on this protein was so recent that papers about it hadn't been published, and was in the process of getting reviewed. Because of this, not only were we unable to receive a plasmid containing the gene, but we couldn't talk about the L. wadei protein in detail.

These legal aspects added an interesting facet to our project. First, we needed to format our experiments in a way that took into account the time necessary for reviews and publications. Second, we had to re-structure the way we described our project so that we didn't talk about L. wadei Cas13a explicitly. In fact, the only reason we can mention it now is that the Zhang Lab recently published their paper on the protein. Finally, it raised the discussion about an important part of biology that many don't always associate with research: patents. We reached out to MIT's Technology Licensing Office to better understand the steps that need to be taken before a new concept can be mentioned and used by others.

Unfortunately, the timeline for paper reviews and iGEM didn't line up, and we were unable to test L. wadei Cas13a in our system. We did, however, learn about the importance of licensing and collaboration.

Burge Lab

Members of our team met with researchers from the Burge Lab to get some advice for our SpliceMIT program. The lab already offers models that will return specific splicing elements present in a given exon sequences that a user inputs. We hoped to apply concepts used in these models to our program. We discussed how the location of binding for the gRNA would have the biggest effect on splicing, and therefore affects what kind of guides the program would return to the user.

Other important aspects discussed include the factors that affect a guide's ability to bind and the strength of such binding. It was from these experts that we learned what to consider for Splice MIT's parameters, as well as how to quantify the "best" guide based on a scoring and ranking system.

CHOP CHOP

CHOP CHOP is a web tool that generates target sites for CRISPR/Cas9, CRISPR/Cpf1 or TALEN-directed mutagenesis. After speaking with developers of the software, we incorporated advice they gave into Splice MIT. Such advice included thoughts on how annotating introns, specifically how to get target sites to appear on introns. They also suggested using a ranking system based on activity scores, complementarity, and off-target effects.

Langer Lab

When thinking about using our system in therapeutics, we knew an important aspect to consider was how the system would be delivered. After some research, we examined solid lipid nanoparticles (SLN) as a potential vehicle. To better understand how SLN worked, we spoke with Dr. Hao Yin from the Langer Lab. Dr. Hao explained that SLNs work best in living systems rather than the HEK cells we intended to transfect. Furthermore, if we were to demonstrate successful proof of concept in our cells, the next step could be to use SLNs. Because of his advice, we did not use SLNs to transfer our system into cells; however, his input was a deciding element for the scope of our project. We worked toward demonstrating that our system could control exclusion of exons so as a possible next step, SLNs could be tested and the therapeutic capability of Cas13a and Ms2 could be explored.