The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is an exciting tool for synthetic biologists because it can target and edit genomes with unprecedented specificity. The Cas9 Protein binds to a PAM site (NGG), attempts to match its sgRNA target sequence to the adjacent DNA strand and cleaves the DNA if it finds a match. Since its popularization, CRISPR papers have flooded major journals and many iGEM teams have worked to improve its use as a genome editing tool.
The 2015 Waterloo team is taking a three-pronged approach to expand upon previous research for wider, more efficient, and more flexible use of CRISPR-Cas9: easy testing of sgRNA designs with our Simple sgRNA Exchange design, applying research on new mutations in Cas9's PAM-interacting domain to enable Cas9 PAM Flexibility and applying our work as a CRISPR Plant Defense protecting A. thaliana against infection by Cauliflower Mosaic Virus.
Simple sgRNA Exchange
We aim to make target selection and guide sequence replacement physically easier in the lab. To target a DNA sequence, a single guide RNA (sgRNA) is used to identify a match . We modify the sgRNA secondary structure analyzed by Briner et al. to contain a restriction site. This makes it possible to swap out a 20 base pair section of the sgRNA sequences, instead of synthesizing new targets from scratch, so there is no need to re-synthesize and re-clone the entire sgRNA sequence for each new target we'd like to test. We hope our design reduces the turnaround time for using CRISPR to target different sequences.
Cas9 PAM Flexibility
By building on recent papers, such as , we are trying to make Cas9’s binding to a protospacer adjacent motif (PAM) site more flexible. The standard S. pyogenes type II CRISPR-Cas9 binds to a PAM of length 3, namely NGG . While this is fairly general, requiring that this sequence be next to the target sequence limits where Cas9 can cut. We have produced a model that suggests Cas9 variants and their preferred PAM sites, and we attempted to demonstrate this model’s validity. The overarching goal is to create Cas9 variants that will bind to any desired PAM site, and we hope to take some significant steps forward to make that goal more achievable and directed.
CRISPR Plant Defense
Finally, CRISPR originated as a viral defence mechanism for bacteria, for specific and targeted immunity . Multi-cellular organisms have developed their own defenses to achieve this goal, but from our research it appears that groups have not publicly attempted to introduce CRISPR as an antiviral mechanism in multicellular organisms. Our team is attempting to use the CRISPR system in plants to discover whether it can defend against a class of double-stranded DNA viruses.