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!
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.
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.
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
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.