The aim of the modelling section was to create an integrated, kinetic model of our Cas9-guided chloroplast transformation mechanism. This model can be used to understand the internal workings of our proposed transformation method, and to determine whether it is viable and genuinely superior to existing methods. It also has practical use, in determining the expected timescales for homoplasmy (and thus when re-plating and selection could plausibly begin). The model is fully documented, and the code is available online and thoroughly commented. It could also be adapted not just to predict homoplasmy times in the chloroplasts of other organisms, but to yield useful information about the kinetics of Cas9-driven genetic transformation in any organism, encompassing the kinetics of:

• Cas9-gRNA active complex formation
• Cas9 cleavage on the chloroplast genome (for both on- and off-target sites)
• integration of the gene of interest, via homologous recombination

PREVIOUS MODELS

Kinetic modelling of CRISPR/Cas9 action has been attempted by several iGEM teams in the past. However, past iGEM models have largely been geared towards using dCas9, a modified Cas9 molecule with no cleavage activity, to form genetic circuits. To the best of our knowledge, no past iGEM project has included a freely available, integrated model of Cas9-induced cleavage or homologous recombination. The Salis lab at Penn State University, however, have recently published a complete, biophysical model of CRISPR/Cas9 cleavage activity [1], which was a major point of reference for our modelling.

MODEL FORMULATION

1. Gene expression

For active Cas9 cleavage, a gRNA template must be transcribed, the Cas9 protein transcribed and translated, and a gRNA-Cas9 complex formed. In our proposed chloroplast transformation method, these are both expressed from the same “driver” cassette, introduced into the chloroplast by a transformation method such as biolistic transformation with a gene gun, but under individual promoters (the psaA promoter for the gRNA, and atpA promoter for Cas9). The Cas9 and gRNA then diffuse randomly through the chloroplast until they encounter one another, at which point they form a Cas9:gRNA complex. A further isomerisation reaction must then take place for this complex to become capable of cleavage. This process is modelled in the following 5 equations:

Where:

• N_gRNA is the number of gRNA strands in a single chloroplast
• N_{Cas9 mRNA} is the number of mRNA strands coding for Cas9 in a single chloroplast
• integration of the gene of interest, via homologous recombination

A set of diagnostic colony PCR were conducted but were inconclusive. The first round was used by directly picking a colony and restreaking on a new plate. The primers were designed to anneal to the backbone and the inserted gene of interest, as this was indicative of both the presence of the backbone as well as the gene of interest. The gel showed a smear which we thought was due to an excess of genetic material. The PCR was repeated with the same conditions, but this time the picked colony was diluted into 10 μL of water before adding 1 μL to the PCR reaction mix. Further, a positive control for the backbones was included. Yet, still the results were inconclusive and needed more work trouble-shooting.

This first intent shows to a certain degree, the potential of parts to make modular constructs to transform chloroplasts in plant organisms, widening the scope of the iGEM competition for new and exciting ideas. Requiring optimization the protocol can be further improved by subsequent teams, as plant synthetic biology grows within iGEM. Future work also involves the use of the DIY open-source biolistics gene gun we have designed and built over the summer.

Cas9 Modelling

We managed to write a code that would enable anyone to predict the time taken to achieve homoplasmy using the appropriate model for Cas9 activity. This program is also designed to be flexible in the sense that various input parameters can be changed to fit the parameters of your experiment. For more information, please read a complete documentation of the program under the 'Modelling' page.