Difference between revisions of "Team:Michigan/Description"

Project Description

We started our design process by setting our team’s design goals for the project:

• The Testing Model’s Cas9s had to bind to the same target sequence via their gRNA in order to build a competitive system.
• The Testing Model needed two different Cas9s that would show different affinities and efficiencies, allowing one Cas9 to outcompete the other.
• The Testing Model needed to be measured in an easy and effective way to allow other iGem teams to replicate and use the model for further modification of CRISPR Cas9.
• The Testing Model needed an active Cas9 to degrade the reporter plasmid and a dead Cas9 to protect the reporter plasmid.
• The Testing Model needed to be inducible for the control of Cas9 expression and thus measurement.

We then reviewed literature and the iGEM registry in search of parts that would help us accomplish our goals. Our initial design called for three plasmids, two with different Cas9s and a reporter plasmid for measurement. After consulting Dr. Zhang, we were able to use her Streptococcus pyogenes CRISPR Cas9 (SpCas9) in an inducible system. After consulting the iGEM registry, we could not find a Streptococcus pyogenes Cas9 with any functional modifications that would change binding efficiencies or affinities. In our efforts to find a Cas9, we came across Streptococcus aureus Cas9 (SaCas9) that is known to have function differences (Friedland et al. 2015). However, because there was no SaCas9 in stock, we synthesized SaCas9 through IDT in a similar inducible model as the Zhang SpCas9. To test if this design met our goals, we devised the experiments outlined in detail on the Experiments page.
SpCas9 and SaCas9 are both proteins frequently used for genetic engineering purposes, particularly as part of the CRISPR-Cas9 platform. The easiest distinction to be made is that the proteins are from different species of bacteria (the former from Streptococcus pyogenes and the latter from Staphylococcus aureus), corresponding with their abbreviated names. Although they are comparable in terms of binding/cutting efficiency, SpCas9 and SaCas9 display several key differences in functionality and characterization which may lend them suitable for certain applications. For instance, SpCas9 is slightly larger at 1368 amino acids (as opposed to SaCas9 with 1053 amino acids), requiring its coding sequence to also be longer. This bp length difference, while minor, may impact transformation efficiency if the overall plasmid is too large. The most important of these differences in terms of practical purposes however is the fact that the Cas9 varieties bind different PAM sequences, with SpCas9 recognizing *NGG and SaCas9 recognizing *NGGRRT. This effectively lessens the probability of off-target cuts in SaCas9 in comparison to SpCas9, due to the greater number of complementary base-pairs needed to bind the PI domain of Cas9 to its PAM sequence. In relation to experiments performed, this necessitated the creation of a PAM sequence that would be compatible with both Cas9 types, although this was not particularly difficult due to the first 3 bases being shared between both PAMs.
In order for the both Cas9s to bind to the same target sequence, we needed each PAM sequence to be represented downstream of the sequence. The PAM sequence for Streptococcus pyogenes Cas9 is *NGG and the PAM sequence for Streptococcus aureus Cas9 is *NGGRRT thus we decided on a sequence that was necessary for both Cas9s to bind - TGGGAT. The guide RNA needed to bind to the DNA (crRNA) that is transcribed is the same for both species of Cas9, but the tracrRNA (gRNA scaffold for Cas9/gRNA interaction) is specific to the species of Cas9.
In designing the gRNA, it is important to note which sequence needs to be transcribed for use by the Cas9. In order for the gRNA/Cas9 to recognize and bind to the target DNA, the crRNA must be the same sequence just upstream of the PAM sequence, thus it binds to the complementary DNA of the PAM sequence.

Proof of Concept

The application of synthetic biology and engineering principles to the Cas9 enzyme could have the potential to expand its use as a powerful tool with a wide range of applications. A wide range of papers have been published in recent years describing various modifications to the Cas9 enzyme. Many different methods were used in these papers to measure the changes to the enzyme’s functionality. This makes it tough to compare Cas9 modifications between papers. We saw a need for a standardized testing system so that researchers can more easily understand how their modifications compare to others. With this in mind, we set out to create a testing platform that will allow other researchers to rapidly and effectively test the modifications they are making to the enzymes. This goal led to the idea of developing a competition based assay where there is a definitive winner of each target binding site between two competing Cas9 enzymes. The Guard Assassin CRISPR/Cas9 Assay (GACCA) allows for this direct competition through the use of a three plasmid system. The first plasmid serves as the target plasmid with a RFP reporter downstream from a target site that both Cas9 enzymes will be attempting to bind. The second plasmid will contain one of the Cas9 enzymes with normal nuclease activity (i.e. able to cut the DNA normally) while the third plasmid contains a dCas9 system with deactivated nuclease activity (i.e. able to bind but not cleave the DNA). The core principle is that the dCas9 will be able to bind to the target sequence before the regular Cas9 if its binding affinity or searching ability is better. If this is the case, the plasmid should be protected from cleavage and degradation of the target plasmid. Then the roles of “Assassin” and “Guard” would be flipped so that the Cas9 that was active previously would now be deactivated and vice versa.
We had considerable difficulty cloning our Cas9 sequences into the intended vectors and thus we were not able to generate any data on the actual performance of this system given the short time scale on which we were working. Given more time, we would establish controls that demonstrate baseline fluorescence, the ability of nuclease active Cas9 to cleave the target plasmid, and the ability of deactivated Cas9 (dCas9) to bind but not cut the target sequence. Once these controls are established and standardized, we would then move into the competition experiments. Additionally, we developed a simple computation model in MATLAB that show the potential effect of enzyme modification on the amount of each complex that is able to form. We intend on fitting this model to data that we generate in the future and showing other teams how they can do the same for their own experiments as they test their Cas9 designs.