Team:NTU-Singapore
Our CRISPRy Adventure! :)
This summer we have set out for an adventure to improve the CRISPR/Cas9 system for better application in mammalian cells. This is our iGEMventure!
Abstract
Our project this summer aims to improve the CRISPR/Cas9 Tool Box for genome engineering and regulation. The Cas9/Cpf1 protein is widely adopted for it has easy programmability to generate a targeted double-strand break. The cell’s repair machinery would then mend the breaks which allows us to make edits to the DNA. To date, several question regarding this technology have been left unanswered. Firstly, despite the variety of Cas9/Cpf1 proteins discovered, we still do not know the efficiency of each protein when compared to each other. Furthermore, the cutting efficiency for different cut sites varies. The popularity of this technology come from it's ability to enhance the rate of knock-ins. However, its efficiency still remains to be improved as efficiencies varied between target sites.
The goal for our team is to make a thorough comparison among different Cas9 proteins and improve it's efficiency in genomic editing.
Project Evaluation
Although several Cas9/Cpf1 have been discovered for gene editing, a thorough comparison of Cas9/Cpf1 from other species are relatively unexplored. Thus, we set out to evaluate and compare the efficiency of CRISPR system across different species.
Project Truncation
Like the iPhone in the smartphone market, competition to produce the slimmest phone always pushes the limit of technology. For Cas9, the same competition applies. We need a smaller Cas9/dCas9 for packaging into the AAV vector. See how we produce dCas9 Air in our Proof of Concept page
Project Evolution
With results from our survey indicating the acceptance of the public towards the CRISPR/Cas9 teachnology, our team was determined to improve the cutting efficiency of SpCas9. By random mutagenesis method, mutant libraries of SpCas9 variants carrying mutated HNH, RuvCII and RuvCIII nuclease domains are generated for downstream selection and characterisation of its efficiency.
Our Great Big Achievements!
Gold
1.Expand on your silver medal activity by demonstrating how you have integrated the investigated issues into the design and/or execution of your project.
2. Improve the function OR characterization of an existing iGEM project (that your team did not originally create) and display your achievement on your wiki.
We have successfully made truncations on SP-dCas9 which previously have been attempted by Team Freiburg 2013. Check out our Description page
3. Demonstrate a functional proof of concept of your project. (biological materials may not be taken outside the lab).
Our predicted truncations are able to activate a ZsGreen reporter with 2 Cas9 binding sites(BBa_K2130003). On the other hand, we have measured 5 different species of Cas9/Cpf1 in various ways. For project Evolution, the evolution assay have generated mutants that have evolved and are selected over WT Sp-Cas9. Check out our Proof of Concept page to discover the secretes of Cas9.
4.Show your project working under real-world conditions. To achieve this criterion, you should demonstrate your whole system, or a functional proof of concept working under simulated conditions in the lab (biological materials may not be taken outside the lab).
Our truncated Sp-dCas9 that have been shown to work in our proof of concept experiment is able to activate endogenous genes. Read more in our Demonstrate page
Silver
1. Validate that something you created (art & design, hardware, software, etc) performs its intended function. Provide thorough documentation of this validation on your team wiki.
We validated that our Cas9/Cpf1 constructs are able to perform as expected before proceeding to make our measurements. Visit Proof of Concept for more info
2. Convince the judges you have helped any registered iGEM team from high school, a different track, another university, or another institution in a significant way by, for example, mentoring a new team, characterizing a part, debugging a construct, modeling/simulating their system or helping validate a software/hardware solution to a synbio problem.
We have helped out Team Macquarie-Australia and Team NUS_Singapore on several experiments. Find out more at our Collaboration page
3. iGEM projects involve important questions beyond the lab bench, for example relating to (but not limited to) ethics, sustainability, social justice, safety, security, and intellectual property rights. Demonstrate how your team has identified, investigated, and addressed one or more of these issues in the context of your project. Your activity could center around education, public engagement, public policy issues, public perception, or other activities (see the human practices hub for more information and examples of previous teams' exemplary work).
We have created art-installations and 3D models regarding CRISPR for a nation-wide exhibition to educate the public about CRISPR/Cas9 technology. Check out our Human Practice page
Bronze
1. Register for iGEM, have a great summer, and attend the Giant Jamboree.
It was super great!
2. Meet all deliverables on the Requirements page (section 3), except those that specifically mention parts.
3. Create a page on your team wiki with clear attribution of each aspect of your project. This page must clearly attribute work done by the students and distinguish it from work done by others, including host labs, advisors, instructors, sponsors, professional website designers, artists, and commercial services.
4. Document at least one new substantial contribution to the iGEM community that showcases a project made with BioBricks. This contribution should be equivalent in difficulty to making and submitting a BioBrick part.
