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.